Proteopedia - User contributions [en]

Malaria Parasite Plasmodium falciparum Erythrocyte Binding Antigen 140

2014-10-13T03:09:25Z

Emily Lum:

== Introduction ==
In 2010, malaria caused over 650,000 deaths.<ref name="World Health Organization">http://www.who.int/mediacentre/factsheets/fs094/en/index.html</ref> While this disease is both preventable and curable, many of those that live in areas where the disease is endemic do not have access to such resources. Although there is a vaccine currently undergoing clinical trials, results are not expected until 2014.<ref name="World Health Organization">http://www.who.int/mediacentre/factsheets/fs094/en/index.html</ref> The most recent vaccine research has focused on understanding a group of proteins in the erythrocyte-binding ligand (EBL) family. These proteins, which are found in the ''Plasmodium'' species, facilitate entry into erythrocytes during malarial infection by creating tight junctions between the host erythrocytes and parasite membranes. <ref name="Lin">PMID: 22989878</ref> There are four family members: erythrocyte-binding antigen 175 (PfEBA-175), erythrocyte-binding ligand 1 (PfEBL-1), erythrocyte-binding antigen 140 (PfEBA-140), and erythrocyte-binding antigen 181 (PfEBA-181).<ref name="Lin">PMID: 22989878</ref> PfEBA-140 binds glycophorin C on host erythrocytes,<ref name="Lin">PMID: 22989878</ref> which helps maintain erythrocyte shape and regulates membrane material properties.<ref name="Glycophorin C">http://en.wikipedia.org/wiki/Glycophorin_C</ref> Understanding the mechanism by which PfEBA-140 recognizes and engages glycophorin C on erythrocytes may lead to the future development of a new malaria vaccine, as it has been found to be immunogenic.<ref name="Ford">PMID: 17984363</ref>

==General Structure==
<Structure load='4GF2' size='400' frame='true' align='left' caption='Crystal Structure of PfEBA-140 (PDB entry [[4gf2]])' scene=''/>
[[Image:SubdomainsF1F2.png | thumb | 300px | alt=text | Subdomains of F1 and F2]]
[[Image:IndividualSubdomains.png | thumb | 300px | alt=text | Subdomains of F1 and F2]]
The EBL family members, including PfEBA-140, are made up of two regions, region II (RII) (shown to the right) and region VI.<ref name="Lin">PMID: 22989878</ref> Region II is responsible for receptor binding in all EBL family members.<ref name="Lin">PMID: 22989878</ref> RII is composed of <scene name='PfEBA-140/Rii/1'>two Duffy binding-like (DBL) domains</scene>, F1 (purple) and F2 (teal).<ref name="Lin">PMID: 22989878</ref> These two DBL domains are connected by a <scene name='PfEBA-140/Helical_linker/2'>short helical linker</scene>.<ref name="Lin">PMID: 22989878</ref> The DBL protein fold is unique to the ''Plasmodium'' species. Not only does it have the ability to recognize and bind many erythrocyte cell receptors, but it also mediates microvasculature adherence of infected erythrocytes by erythrocyte membrane protein 1 (PfEMP1).<ref name="Lin">PMID: 22989878</ref> Each DBL domain is composed of three subdomains, illustrated in the pictures to the right.

In the top image, the subdomains S1, S2, and S3 of each of the F1 and F2 domains, as well as the helical linker, are illustrated.<ref name="Lin">PMID: 22989878</ref>
In the bottom image, structures of the individual subdomains are illustrated.<ref name="Lin">PMID: 22989878</ref>

The colors are the same in both images: F1 subdomain 1 is shown in bronze, subdomain 2 in orange, subdomain 3 in dark orange; F2 subdomain 1 is shown in dark blue, subdomain 2 in blue, and subdomain 3 in light blue.

 
 
 

==Binding of RII PfEBA-140==
<Structure load='4gf2' size='400' frame='true' align='left' caption='Binding of PfEBA-140 (PDB entry [[4gf2]])' scene=''/>
===Requirement of Both DBL Domains===
[[Image:Binding.png | thumb | 100px | alt=text | Erythrocyte Binding of Full Length and Individual RII Domains]]Since the DBL domains of RII are highly conserved, the ability of the individual DBL domains to bind erythrocytes was tested. To do this, constructs containing the full-length RII PfEBA-140 and each individual DBL domain were tested using a rosetting assay. Both brightfield microscopy and green fluorescence protein (GFP) were used to visualize erythrocyte binding. In the upper panel, a construct expressing only GFP was used as a control. The lower panel of Figure A illustrates the extensive erythrocyte binding of the full length RII construct. In Figure B, the lack of black dots in the brightfield microscopy and the smaller quantity of green fluorescence illustrates that F1 and F2 are unable to independently bind erythrocytes. This result suggests that both domains equally participate in engaging erythrocytes.<ref name="Lin">PMID: 22989878</ref>
===Basic Patch===
[[Image:Basic Patch.png | thumb | 100px | alt=text | Erythrocyte Binding of Full Length and Individual RII Domains]]
Since the two DBL domains bind acidic GPC, there must be a number of basic charges that interact with the acidic GPC residues. This idea is supported by visualizing the <scene name='PfEBA-140/Basic_patch/1'>positive and negative residues</scene> found in RII PfEBA-140. The positive side chain residues are bue while the negative side chain residues are red. On the side shown, there are a number of residues with positive side chains, creating a basic patch. This basic patch, which forms an arch spanning the two DBL domains, is thought to interact with the acidic GPC, because if the molecule is flipped 180°, there is no concentration of positive charges. The basic patch may be better visualized in the picture to the right.<ref name="Lin">PMID: 22989878</ref>
To test whether this basic patch was indeed responsible for erythrocyte binding, two residues in each DBL domain were chosen and mutated to Alanine. In F1, these residues were <scene name='PfEBA-140/Asp247/1'>Asp247</scene> and <scene name='PfEBA-140/Arg254/1'>Arg254</scene>. In F2, these residues were <scene name='PfEBA-140/Arg485/1'>Arg485</scene> and <scene name='PfEBA-140/Asp554/2'>Asp554</scene>. When these four residues were individually mutated to Alanine, erythrocyte binding was heavily interrupted. This can be observed in the figure to the right.<ref name="Lin">PMID: 22989878</ref>[[Image:DecreasedBinding.png | thumb | 100px | alt=text | Mutation of Asp247, Arg254, Arg485, and Asp554]] The decreased erythrocyte binding confirms that these <scene name='PfEBA-140/Four/1'>four residues</scene> found in the basic patch of RII PfEBA-140 are crucial to erythrocyte binding. As a control, residues on the opposite side of RII PfEBA-140 were mutated to Alanine. These residues were Asn394 and Glu605. Mutation of these two residues had no effect on erythrocyte binding.<ref name="Lin">PMID: 22989878</ref>[[Image:FullBinding.png | thumb | 100px | alt=text | Mutation of Asn394 and Glu605]] These results fortify the finding that erythrocyte binding takes place in the basic patch discussed earlier.

 
 
 
 
 
 

==Structural Differences in PfEBA-140 from other EBL Ligands==
<Structure load='4gf2' size='400' frame='true' align='left' caption='Structural Characteristics of PfEBA-140 (PDB entry [[4gf2]]) leading to Binding Specificity' scene=''/>
There are three unique structural elements that set RII PfEBA-140 apart from other EBL ligands. These structures are the likely reason behind RII PfEBA-140 binding GPC so specifically.
===Disulfide Bonds===
[[Image:ModifiedPattern.png | thumb | 300px | alt=text | Disulfide Bonding Patterns]]
There are twenty-six <scene name='PfEBA-140/Cysteines/1'>cysteines</scene> in RII PfEBA-140, all of which are involved in <scene name='PfEBA-140/Disulfidebonds/1'>disulfide bonding</scene> (disulfide bonds shown in black).<ref name="Lin">PMID: 22989878</ref> In RII PfEBA-140, two modified disulfide patterns were found to be different than in RII PfEBA-175, RII PvDBP, and RII PkDBP. In the latter three RII domains, there is a disulfide linkage between Cys7 and Cys9 and between Cys8 and Cys12. In RII PfEBA-140, however, Cys7 is disulfide bonded to Cys8, and Cys9 is disulfide bonded to Cys12.<ref name="Lin">PMID: 22989878</ref>

===Alpha Helix Structure===
Another difference between RII PfEBA-140 and other EBL ligands can be found in the F2 domain. In EBL ligands of other ''Plasmodium'' species, there are β-fingers with either long or short loops which may or may not be important for receptor binding.<ref name="Lin">PMID: 22989878</ref> While this β-finger is found in F1 of PfEBA-140, it is not found in F2, as it is replaced with an <scene name='PfEBA-140/Alphabeta/1'>α-helix</scene>.<ref name="Lin">PMID: 22989878</ref>
===Lack of Kink in F1===
A third difference between RII PfEBA-140 and other EBL ligands is the lack of a glycine residue in F1. This glycine is present in F1 and F2 of PfEBA-175, PvDBP, PkDBP-α, and F2 PfEBA-140. In F2 PfEBA-140, this glycine is residue 627.<ref name="Lin">PMID: 22989878</ref> In these EBL ligands there is a kink due to glycine's flexibility. If this glycine residue were present in F1 of PfEBA-140, it would result in a kink in subdomain 3.<ref name="Lin">PMID: 22989878</ref> This kink would create a hinge angle between the two DBL domains that would allow for dimerization of PfEBA-140 upon erythrocyte binding. For this reason, PfEBA-140 is believed to engage erythrocytes as a monomer, unlike other EBL ligands, which bind erythrocytes as a dimer.
 
 
These three structural differences are believed to be responsible for the specificity of RII PfEBA-140.

==References==
<references />

Malaria Parasite Plasmodium falciparum Erythrocyte Binding Antigen 140

2012-11-24T22:41:38Z

Emily Lum: /* Structural Differences in PfEBA-140 from other EBL Ligands */

== Introduction ==
In 2010, malaria caused over 650,000 deaths.<ref name="World Health Organization">http://www.who.int/mediacentre/factsheets/fs094/en/index.html</ref> While this disease is both preventable and curable, many of those that live in areas where the disease is endemic do not have access to such resources. Although there is a vaccine currently undergoing clinical trials, results are not expected until 2014.<ref name="World Health Organization">http://www.who.int/mediacentre/factsheets/fs094/en/index.html</ref> The most recent vaccine research has focused on understanding a group of proteins in the erythrocyte-binding ligand (EBL) family. These proteins, which are found in the ''Plasmodium'' species, facilitate entry into erythrocytes during malarial infection by creating tight junctions between the host erythrocytes and parasite membranes. <ref name="Lin">PMID: 22989878</ref> There are four family members: erythrocyte-binding antigen 175 (PfEBA-175), erythrocyte-binding ligand 1 (PfEBL-1), erythrocyte-binding antigen 140 (PfEBA-140), and erythrocyte-binding antigen 181 (PfEBA-181).<ref name="Lin">PMID: 22989878</ref> PfEBA-140 binds glycophorin C on host erythrocytes,<ref name="Lin">PMID: 22989878</ref> which helps maintain erythrocyte shape and regulates membrane material properties.<ref name="Glycophorin C">http://en.wikipedia.org/wiki/Glycophorin_C</ref> Understanding the mechanism by which PfEBA-140 recognizes and engages glycophorin C on erythrocytes may lead to the future development of a new malaria vaccine, as it has been found to be immunogenic.<ref name="Ford">PMID: 17984363</ref>

==General Structure==
<Structure load='4GF2' size='400' frame='true' align='left' caption='Crystal Structure of PfEBA-140 (PDB entry [[4gf2]])' scene=''/>
[[Image:SubdomainsF1F2.png | thumb | 300px | alt=text | Subdomains of F1 and F2]]
[[Image:IndividualSubdomains.png | thumb | 300px | alt=text | Subdomains of F1 and F2]]
The EBL family members, including PfEBA-140, are made up of two regions, region II (RII) (shown to the right) and region VI.<ref name="Lin">PMID: 22989878</ref> Region II is responsible for receptor binding in all EBL family members.<ref name="Lin">PMID: 22989878</ref> RII is composed of <scene name='PfEBA-140/Rii/1'>two Duffy binding-like (DBL) domains</scene>, F1 (purple) and F2 (teal).<ref name="Lin">PMID: 22989878</ref> These two DBL domains are connected by a <scene name='PfEBA-140/Helical_linker/2'>short helical linker</scene>.<ref name="Lin">PMID: 22989878</ref> The DBL protein fold is unique to the ''Plasmodium'' species. Not only does it have the ability to recognize and bind many erythrocyte cell receptors, but it also mediates microvasculature adherence of infected erythrocytes by erythrocyte membrane protein 1 (PfEMP1).<ref name="Lin">PMID: 22989878</ref> Each DBL domain is composed of three subdomains, illustrated in the pictures to the right.

In the top image, the subdomains S1, S2, and S3 of each of the F1 and F2 domains, as well as the helical linker, are illustrated.<ref name="Lin">PMID: 22989878</ref>
In the bottom image, structures of the individual subdomains are illustrated.<ref name="Lin">PMID: 22989878</ref>

The colors are the same in both images: F1 subdomain 1 is shown in bronze, subdomain 2 in orange, subdomain 3 in dark orange; F2 subdomain 1 is shown in dark blue, subdomain 2 in blue, and subdomain 3 in light blue.

==Binding of RII PfEBA-140==
<Structure load='4gf2' size='400' frame='true' align='left' caption='Binding of PfEBA-140 (PDB entry [[4gf2]])' scene=''/>
===Requirement of Both DBL Domains===
[[Image:Binding.png | thumb | 100px | alt=text | Erythrocyte Binding of Full Length and Individual RII Domains]]Since the DBL domains of RII are highly conserved, the ability of the individual DBL domains to bind erythrocytes was tested. To do this, constructs containing the full-length RII PfEBA-140 and each individual DBL domain were tested using a rosetting assay. Both brightfield microscopy and green fluorescence protein (GFP) were used to visualize erythrocyte binding. In the upper panel, a construct expressing only GFP was used as a control. The lower panel of Figure A illustrates the extensive erythrocyte binding of the full length RII construct. In Figure B, the lack of black dots in the brightfield microscopy and the smaller quantity of green fluorescence illustrates that F1 and F2 are unable to independently bind erythrocytes. This result suggests that both domains equally participate in engaging erythrocytes.<ref name="Lin">PMID: 22989878</ref>
===Basic Patch===
[[Image:Basic Patch.png | thumb | 100px | alt=text | Erythrocyte Binding of Full Length and Individual RII Domains]]
Since the two DBL domains bind acidic GPC, there must be a number of basic charges that interact with the acidic GPC residues. This idea is supported by visualizing the <scene name='PfEBA-140/Basic_patch/1'>positive and negative residues</scene> found in RII PfEBA-140. The positive side chain residues are bue while the negative side chain residues are red. On the side shown, there are a number of residues with positive side chains, creating a basic patch. This basic patch, which forms an arch spanning the two DBL domains, is thought to interact with the acidic GPC, because if the molecule is flipped 180°, there is no concentration of positive charges. The basic patch may be better visualized in the picture to the right.<ref name="Lin">PMID: 22989878</ref>
To test whether this basic patch was indeed responsible for erythrocyte binding, two residues in each DBL domain were chosen and mutated to Alanine. In F1, these residues were <scene name='PfEBA-140/Asp247/1'>Asp247</scene> and <scene name='PfEBA-140/Arg254/1'>Arg254</scene>. In F2, these residues were <scene name='PfEBA-140/Arg485/1'>Arg485</scene> and <scene name='PfEBA-140/Asp554/2'>Asp554</scene>. When these four residues were individually mutated to Alanine, erythrocyte binding was heavily interrupted. This can be observed in the figure to the right.<ref name="Lin">PMID: 22989878</ref>[[Image:DecreasedBinding.png | thumb | 100px | alt=text | Mutation of Asp247, Arg254, Arg485, and Asp554]] The decreased erythrocyte binding confirms that these <scene name='PfEBA-140/Four/1'>four residues</scene> found in the basic patch of RII PfEBA-140 are crucial to erythrocyte binding. As a control, residues on the opposite side of RII PfEBA-140 were mutated to Alanine. These residues were Asn394 and Glu605. Mutation of these two residues had no effect on erythrocyte binding.<ref name="Lin">PMID: 22989878</ref>[[Image:FullBinding.png | thumb | 100px | alt=text | Mutation of Asn394 and Glu605]] These results fortify the finding that erythrocyte binding takes place in the basic patch discussed earlier.

 
 
 
 
 

==Structural Differences in PfEBA-140 from other EBL Ligands==
<Structure load='4gf2' size='400' frame='true' align='left' caption='Structural Characteristics of PfEBA-140 (PDB entry [[4gf2]]) leading to Binding Specificity' scene=''/>
There are three unique structural elements that set RII PfEBA-140 apart from other EBL ligands. These structures are the likely reason behind RII PfEBA-140 binding GPC so specifically.
===Disulfide Bonds===
[[Image:ModifiedPattern.png | thumb | 300px | alt=text | Disulfide Bonding Patterns]]
There are twenty-six <scene name='PfEBA-140/Cysteines/1'>cysteines</scene> in RII PfEBA-140, all of which are involved in <scene name='PfEBA-140/Disulfidebonds/1'>disulfide bonding</scene> (disulfide bonds shown in black).<ref name="Lin">PMID: 22989878</ref> In RII PfEBA-140, two modified disulfide patterns were found to be different than in RII PfEBA-175, RII PvDBP, and RII PkDBP. In the latter three RII domains, there is a disulfide linkage between Cys7 and Cys9 and between Cys8 and Cys12. In RII PfEBA-140, however, Cys7 is disulfide bonded to Cys8, and Cys9 is disulfide bonded to Cys12.<ref name="Lin">PMID: 22989878</ref>

===Alpha Helix Structure===
Another difference between RII PfEBA-140 and other EBL ligands can be found in the F2 domain. In EBL ligands of other ''Plasmodium'' species, there are β-fingers with either long or short loops which may or may not be important for receptor binding.<ref name="Lin">PMID: 22989878</ref> While this β-finger is found in F1 of PfEBA-140, it is not found in F2, as it is replaced with an <scene name='PfEBA-140/Alphabeta/1'>α-helix</scene>.<ref name="Lin">PMID: 22989878</ref>
===Lack of Kink in F1===
A third difference between RII PfEBA-140 and other EBL ligands is the lack of a glycine residue in F1. This glycine is present in F1 and F2 of PfEBA-175, PvDBP, PkDBP-α, and F2 PfEBA-140. In F2 PfEBA-140, this glycine is residue 627.<ref name="Lin">PMID: 22989878</ref> In these EBL ligands there is a kink due to glycine's flexibility. If this glycine residue were present in F1 of PfEBA-140, it would result in a kink in subdomain 3.<ref name="Lin">PMID: 22989878</ref> This kink would create a hinge angle between the two DBL domains that would allow for dimerization of PfEBA-140 upon erythrocyte binding. For this reason, PfEBA-140 is believed to engage erythrocytes as a monomer, unlike other EBL ligands, which bind erythrocytes as a dimer.
 
 
These three structural differences are believed to be responsible for the specificity of RII PfEBA-140.

==References==
<references />

Malaria Parasite Plasmodium falciparum Erythrocyte Binding Antigen 140

2012-11-24T22:41:04Z

Emily Lum: /* Structural Differences in PfEBA-140 from other EBL Ligands */

== Introduction ==
In 2010, malaria caused over 650,000 deaths.<ref name="World Health Organization">http://www.who.int/mediacentre/factsheets/fs094/en/index.html</ref> While this disease is both preventable and curable, many of those that live in areas where the disease is endemic do not have access to such resources. Although there is a vaccine currently undergoing clinical trials, results are not expected until 2014.<ref name="World Health Organization">http://www.who.int/mediacentre/factsheets/fs094/en/index.html</ref> The most recent vaccine research has focused on understanding a group of proteins in the erythrocyte-binding ligand (EBL) family. These proteins, which are found in the ''Plasmodium'' species, facilitate entry into erythrocytes during malarial infection by creating tight junctions between the host erythrocytes and parasite membranes. <ref name="Lin">PMID: 22989878</ref> There are four family members: erythrocyte-binding antigen 175 (PfEBA-175), erythrocyte-binding ligand 1 (PfEBL-1), erythrocyte-binding antigen 140 (PfEBA-140), and erythrocyte-binding antigen 181 (PfEBA-181).<ref name="Lin">PMID: 22989878</ref> PfEBA-140 binds glycophorin C on host erythrocytes,<ref name="Lin">PMID: 22989878</ref> which helps maintain erythrocyte shape and regulates membrane material properties.<ref name="Glycophorin C">http://en.wikipedia.org/wiki/Glycophorin_C</ref> Understanding the mechanism by which PfEBA-140 recognizes and engages glycophorin C on erythrocytes may lead to the future development of a new malaria vaccine, as it has been found to be immunogenic.<ref name="Ford">PMID: 17984363</ref>

==General Structure==
<Structure load='4GF2' size='400' frame='true' align='left' caption='Crystal Structure of PfEBA-140 (PDB entry [[4gf2]])' scene=''/>
[[Image:SubdomainsF1F2.png | thumb | 300px | alt=text | Subdomains of F1 and F2]]
[[Image:IndividualSubdomains.png | thumb | 300px | alt=text | Subdomains of F1 and F2]]
The EBL family members, including PfEBA-140, are made up of two regions, region II (RII) (shown to the right) and region VI.<ref name="Lin">PMID: 22989878</ref> Region II is responsible for receptor binding in all EBL family members.<ref name="Lin">PMID: 22989878</ref> RII is composed of <scene name='PfEBA-140/Rii/1'>two Duffy binding-like (DBL) domains</scene>, F1 (purple) and F2 (teal).<ref name="Lin">PMID: 22989878</ref> These two DBL domains are connected by a <scene name='PfEBA-140/Helical_linker/2'>short helical linker</scene>.<ref name="Lin">PMID: 22989878</ref> The DBL protein fold is unique to the ''Plasmodium'' species. Not only does it have the ability to recognize and bind many erythrocyte cell receptors, but it also mediates microvasculature adherence of infected erythrocytes by erythrocyte membrane protein 1 (PfEMP1).<ref name="Lin">PMID: 22989878</ref> Each DBL domain is composed of three subdomains, illustrated in the pictures to the right.

In the top image, the subdomains S1, S2, and S3 of each of the F1 and F2 domains, as well as the helical linker, are illustrated.<ref name="Lin">PMID: 22989878</ref>
In the bottom image, structures of the individual subdomains are illustrated.<ref name="Lin">PMID: 22989878</ref>

The colors are the same in both images: F1 subdomain 1 is shown in bronze, subdomain 2 in orange, subdomain 3 in dark orange; F2 subdomain 1 is shown in dark blue, subdomain 2 in blue, and subdomain 3 in light blue.

==Binding of RII PfEBA-140==
<Structure load='4gf2' size='400' frame='true' align='left' caption='Binding of PfEBA-140 (PDB entry [[4gf2]])' scene=''/>
===Requirement of Both DBL Domains===
[[Image:Binding.png | thumb | 100px | alt=text | Erythrocyte Binding of Full Length and Individual RII Domains]]Since the DBL domains of RII are highly conserved, the ability of the individual DBL domains to bind erythrocytes was tested. To do this, constructs containing the full-length RII PfEBA-140 and each individual DBL domain were tested using a rosetting assay. Both brightfield microscopy and green fluorescence protein (GFP) were used to visualize erythrocyte binding. In the upper panel, a construct expressing only GFP was used as a control. The lower panel of Figure A illustrates the extensive erythrocyte binding of the full length RII construct. In Figure B, the lack of black dots in the brightfield microscopy and the smaller quantity of green fluorescence illustrates that F1 and F2 are unable to independently bind erythrocytes. This result suggests that both domains equally participate in engaging erythrocytes.<ref name="Lin">PMID: 22989878</ref>
===Basic Patch===
[[Image:Basic Patch.png | thumb | 100px | alt=text | Erythrocyte Binding of Full Length and Individual RII Domains]]
Since the two DBL domains bind acidic GPC, there must be a number of basic charges that interact with the acidic GPC residues. This idea is supported by visualizing the <scene name='PfEBA-140/Basic_patch/1'>positive and negative residues</scene> found in RII PfEBA-140. The positive side chain residues are bue while the negative side chain residues are red. On the side shown, there are a number of residues with positive side chains, creating a basic patch. This basic patch, which forms an arch spanning the two DBL domains, is thought to interact with the acidic GPC, because if the molecule is flipped 180°, there is no concentration of positive charges. The basic patch may be better visualized in the picture to the right.<ref name="Lin">PMID: 22989878</ref>
To test whether this basic patch was indeed responsible for erythrocyte binding, two residues in each DBL domain were chosen and mutated to Alanine. In F1, these residues were <scene name='PfEBA-140/Asp247/1'>Asp247</scene> and <scene name='PfEBA-140/Arg254/1'>Arg254</scene>. In F2, these residues were <scene name='PfEBA-140/Arg485/1'>Arg485</scene> and <scene name='PfEBA-140/Asp554/2'>Asp554</scene>. When these four residues were individually mutated to Alanine, erythrocyte binding was heavily interrupted. This can be observed in the figure to the right.<ref name="Lin">PMID: 22989878</ref>[[Image:DecreasedBinding.png | thumb | 100px | alt=text | Mutation of Asp247, Arg254, Arg485, and Asp554]] The decreased erythrocyte binding confirms that these <scene name='PfEBA-140/Four/1'>four residues</scene> found in the basic patch of RII PfEBA-140 are crucial to erythrocyte binding. As a control, residues on the opposite side of RII PfEBA-140 were mutated to Alanine. These residues were Asn394 and Glu605. Mutation of these two residues had no effect on erythrocyte binding.<ref name="Lin">PMID: 22989878</ref>[[Image:FullBinding.png | thumb | 100px | alt=text | Mutation of Asn394 and Glu605]] These results fortify the finding that erythrocyte binding takes place in the basic patch discussed earlier.

 
 
 
 
 

==Structural Differences in PfEBA-140 from other EBL Ligands==
<Structure load='4gf2' size='400' frame='true' align='left' caption='Structure of PfEBA-140 (PDB entry [[4gf2]]) leading to Binding Specificity' scene=''/>
There are three unique structural elements that set RII PfEBA-140 apart from other EBL ligands. These structures are the likely reason behind RII PfEBA-140 binding GPC so specifically.
===Disulfide Bonds===
[[Image:ModifiedPattern.png | thumb | 300px | alt=text | Disulfide Bonding Patterns]]
There are twenty-six <scene name='PfEBA-140/Cysteines/1'>cysteines</scene> in RII PfEBA-140, all of which are involved in <scene name='PfEBA-140/Disulfidebonds/1'>disulfide bonding</scene> (disulfide bonds shown in black).<ref name="Lin">PMID: 22989878</ref> In RII PfEBA-140, two modified disulfide patterns were found to be different than in RII PfEBA-175, RII PvDBP, and RII PkDBP. In the latter three RII domains, there is a disulfide linkage between Cys7 and Cys9 and between Cys8 and Cys12. In RII PfEBA-140, however, Cys7 is disulfide bonded to Cys8, and Cys9 is disulfide bonded to Cys12.<ref name="Lin">PMID: 22989878</ref>

===Alpha Helix Structure===
Another difference between RII PfEBA-140 and other EBL ligands can be found in the F2 domain. In EBL ligands of other ''Plasmodium'' species, there are β-fingers with either long or short loops which may or may not be important for receptor binding.<ref name="Lin">PMID: 22989878</ref> While this β-finger is found in F1 of PfEBA-140, it is not found in F2, as it is replaced with an <scene name='PfEBA-140/Alphabeta/1'>α-helix</scene>.<ref name="Lin">PMID: 22989878</ref>
===Lack of Kink in F1===
A third difference between RII PfEBA-140 and other EBL ligands is the lack of a glycine residue in F1. This glycine is present in F1 and F2 of PfEBA-175, PvDBP, PkDBP-α, and F2 PfEBA-140. In F2 PfEBA-140, this glycine is residue 627.<ref name="Lin">PMID: 22989878</ref> In these EBL ligands there is a kink due to glycine's flexibility. If this glycine residue were present in F1 of PfEBA-140, it would result in a kink in subdomain 3.<ref name="Lin">PMID: 22989878</ref> This kink would create a hinge angle between the two DBL domains that would allow for dimerization of PfEBA-140 upon erythrocyte binding. For this reason, PfEBA-140 is believed to engage erythrocytes as a monomer, unlike other EBL ligands, which bind erythrocytes as a dimer.
 
 
These three structural differences are believed to be responsible for the specificity of RII PfEBA-140.

==References==
<references />

Malaria Parasite Plasmodium falciparum Erythrocyte Binding Antigen 140

2012-11-24T22:39:54Z

Emily Lum: /* Binding of RII PfEBA-140 */

== Introduction ==
In 2010, malaria caused over 650,000 deaths.<ref name="World Health Organization">http://www.who.int/mediacentre/factsheets/fs094/en/index.html</ref> While this disease is both preventable and curable, many of those that live in areas where the disease is endemic do not have access to such resources. Although there is a vaccine currently undergoing clinical trials, results are not expected until 2014.<ref name="World Health Organization">http://www.who.int/mediacentre/factsheets/fs094/en/index.html</ref> The most recent vaccine research has focused on understanding a group of proteins in the erythrocyte-binding ligand (EBL) family. These proteins, which are found in the ''Plasmodium'' species, facilitate entry into erythrocytes during malarial infection by creating tight junctions between the host erythrocytes and parasite membranes. <ref name="Lin">PMID: 22989878</ref> There are four family members: erythrocyte-binding antigen 175 (PfEBA-175), erythrocyte-binding ligand 1 (PfEBL-1), erythrocyte-binding antigen 140 (PfEBA-140), and erythrocyte-binding antigen 181 (PfEBA-181).<ref name="Lin">PMID: 22989878</ref> PfEBA-140 binds glycophorin C on host erythrocytes,<ref name="Lin">PMID: 22989878</ref> which helps maintain erythrocyte shape and regulates membrane material properties.<ref name="Glycophorin C">http://en.wikipedia.org/wiki/Glycophorin_C</ref> Understanding the mechanism by which PfEBA-140 recognizes and engages glycophorin C on erythrocytes may lead to the future development of a new malaria vaccine, as it has been found to be immunogenic.<ref name="Ford">PMID: 17984363</ref>

==General Structure==
<Structure load='4GF2' size='400' frame='true' align='left' caption='Crystal Structure of PfEBA-140 (PDB entry [[4gf2]])' scene=''/>
[[Image:SubdomainsF1F2.png | thumb | 300px | alt=text | Subdomains of F1 and F2]]
[[Image:IndividualSubdomains.png | thumb | 300px | alt=text | Subdomains of F1 and F2]]
The EBL family members, including PfEBA-140, are made up of two regions, region II (RII) (shown to the right) and region VI.<ref name="Lin">PMID: 22989878</ref> Region II is responsible for receptor binding in all EBL family members.<ref name="Lin">PMID: 22989878</ref> RII is composed of <scene name='PfEBA-140/Rii/1'>two Duffy binding-like (DBL) domains</scene>, F1 (purple) and F2 (teal).<ref name="Lin">PMID: 22989878</ref> These two DBL domains are connected by a <scene name='PfEBA-140/Helical_linker/2'>short helical linker</scene>.<ref name="Lin">PMID: 22989878</ref> The DBL protein fold is unique to the ''Plasmodium'' species. Not only does it have the ability to recognize and bind many erythrocyte cell receptors, but it also mediates microvasculature adherence of infected erythrocytes by erythrocyte membrane protein 1 (PfEMP1).<ref name="Lin">PMID: 22989878</ref> Each DBL domain is composed of three subdomains, illustrated in the pictures to the right.

In the top image, the subdomains S1, S2, and S3 of each of the F1 and F2 domains, as well as the helical linker, are illustrated.<ref name="Lin">PMID: 22989878</ref>
In the bottom image, structures of the individual subdomains are illustrated.<ref name="Lin">PMID: 22989878</ref>

The colors are the same in both images: F1 subdomain 1 is shown in bronze, subdomain 2 in orange, subdomain 3 in dark orange; F2 subdomain 1 is shown in dark blue, subdomain 2 in blue, and subdomain 3 in light blue.

==Binding of RII PfEBA-140==
<Structure load='4gf2' size='400' frame='true' align='left' caption='Binding of PfEBA-140 (PDB entry [[4gf2]])' scene=''/>
===Requirement of Both DBL Domains===
[[Image:Binding.png | thumb | 100px | alt=text | Erythrocyte Binding of Full Length and Individual RII Domains]]Since the DBL domains of RII are highly conserved, the ability of the individual DBL domains to bind erythrocytes was tested. To do this, constructs containing the full-length RII PfEBA-140 and each individual DBL domain were tested using a rosetting assay. Both brightfield microscopy and green fluorescence protein (GFP) were used to visualize erythrocyte binding. In the upper panel, a construct expressing only GFP was used as a control. The lower panel of Figure A illustrates the extensive erythrocyte binding of the full length RII construct. In Figure B, the lack of black dots in the brightfield microscopy and the smaller quantity of green fluorescence illustrates that F1 and F2 are unable to independently bind erythrocytes. This result suggests that both domains equally participate in engaging erythrocytes.<ref name="Lin">PMID: 22989878</ref>
===Basic Patch===
[[Image:Basic Patch.png | thumb | 100px | alt=text | Erythrocyte Binding of Full Length and Individual RII Domains]]
Since the two DBL domains bind acidic GPC, there must be a number of basic charges that interact with the acidic GPC residues. This idea is supported by visualizing the <scene name='PfEBA-140/Basic_patch/1'>positive and negative residues</scene> found in RII PfEBA-140. The positive side chain residues are bue while the negative side chain residues are red. On the side shown, there are a number of residues with positive side chains, creating a basic patch. This basic patch, which forms an arch spanning the two DBL domains, is thought to interact with the acidic GPC, because if the molecule is flipped 180°, there is no concentration of positive charges. The basic patch may be better visualized in the picture to the right.<ref name="Lin">PMID: 22989878</ref>
To test whether this basic patch was indeed responsible for erythrocyte binding, two residues in each DBL domain were chosen and mutated to Alanine. In F1, these residues were <scene name='PfEBA-140/Asp247/1'>Asp247</scene> and <scene name='PfEBA-140/Arg254/1'>Arg254</scene>. In F2, these residues were <scene name='PfEBA-140/Arg485/1'>Arg485</scene> and <scene name='PfEBA-140/Asp554/2'>Asp554</scene>. When these four residues were individually mutated to Alanine, erythrocyte binding was heavily interrupted. This can be observed in the figure to the right.<ref name="Lin">PMID: 22989878</ref>[[Image:DecreasedBinding.png | thumb | 100px | alt=text | Mutation of Asp247, Arg254, Arg485, and Asp554]] The decreased erythrocyte binding confirms that these <scene name='PfEBA-140/Four/1'>four residues</scene> found in the basic patch of RII PfEBA-140 are crucial to erythrocyte binding. As a control, residues on the opposite side of RII PfEBA-140 were mutated to Alanine. These residues were Asn394 and Glu605. Mutation of these two residues had no effect on erythrocyte binding.<ref name="Lin">PMID: 22989878</ref>[[Image:FullBinding.png | thumb | 100px | alt=text | Mutation of Asn394 and Glu605]] These results fortify the finding that erythrocyte binding takes place in the basic patch discussed earlier.

 
 
 
 
 

==Structural Differences in PfEBA-140 from other EBL Ligands==
<Structure load='4gf2' size='400' frame='true' align='left' caption='Crystal Structure of PfEBA-140 (PDB entry [[4gf2]])' scene=''/>
There are three unique structural elements that set RII PfEBA-140 apart from other EBL ligands. These structures are the likely reason behind RII PfEBA-140 binding GPC so specifically.
===Disulfide Bonds===
[[Image:ModifiedPattern.png | thumb | 300px | alt=text | Disulfide Bonding Patterns]]
There are twenty-six <scene name='PfEBA-140/Cysteines/1'>cysteines</scene> in RII PfEBA-140, all of which are involved in <scene name='PfEBA-140/Disulfidebonds/1'>disulfide bonding</scene> (disulfide bonds shown in black).<ref name="Lin">PMID: 22989878</ref> In RII PfEBA-140, two modified disulfide patterns were found to be different than in RII PfEBA-175, RII PvDBP, and RII PkDBP. In the latter three RII domains, there is a disulfide linkage between Cys7 and Cys9 and between Cys8 and Cys12. In RII PfEBA-140, however, Cys7 is disulfide bonded to Cys8, and Cys9 is disulfide bonded to Cys12.<ref name="Lin">PMID: 22989878</ref>

===Alpha Helix Structure===
Another difference between RII PfEBA-140 and other EBL ligands can be found in the F2 domain. In EBL ligands of other ''Plasmodium'' species, there are β-fingers with either long or short loops which may or may not be important for receptor binding.<ref name="Lin">PMID: 22989878</ref> While this β-finger is found in F1 of PfEBA-140, it is not found in F2, as it is replaced with an <scene name='PfEBA-140/Alphabeta/1'>α-helix</scene>.<ref name="Lin">PMID: 22989878</ref>
===Lack of Kink in F1===
A third difference between RII PfEBA-140 and other EBL ligands is the lack of a glycine residue in F1. This glycine is present in F1 and F2 of PfEBA-175, PvDBP, PkDBP-α, and F2 PfEBA-140. In F2 PfEBA-140, this glycine is residue 627.<ref name="Lin">PMID: 22989878</ref> In these EBL ligands there is a kink due to glycine's flexibility. If this glycine residue were present in F1 of PfEBA-140, it would result in a kink in subdomain 3.<ref name="Lin">PMID: 22989878</ref> This kink would create a hinge angle between the two DBL domains that would allow for dimerization of PfEBA-140 upon erythrocyte binding. For this reason, PfEBA-140 is believed to engage erythrocytes as a monomer, unlike other EBL ligands, which bind erythrocytes as a dimer.
 
 
These three structural differences are believed to be responsible for the specificity of RII PfEBA-140.

==References==
<references />

Malaria Parasite Plasmodium falciparum Erythrocyte Binding Antigen 140

2012-11-24T22:32:12Z

Emily Lum: /* Structural Differences in PfEBA-140 from other EBL Ligands */

== Introduction ==
In 2010, malaria caused over 650,000 deaths.<ref name="World Health Organization">http://www.who.int/mediacentre/factsheets/fs094/en/index.html</ref> While this disease is both preventable and curable, many of those that live in areas where the disease is endemic do not have access to such resources. Although there is a vaccine currently undergoing clinical trials, results are not expected until 2014.<ref name="World Health Organization">http://www.who.int/mediacentre/factsheets/fs094/en/index.html</ref> The most recent vaccine research has focused on understanding a group of proteins in the erythrocyte-binding ligand (EBL) family. These proteins, which are found in the ''Plasmodium'' species, facilitate entry into erythrocytes during malarial infection by creating tight junctions between the host erythrocytes and parasite membranes. <ref name="Lin">PMID: 22989878</ref> There are four family members: erythrocyte-binding antigen 175 (PfEBA-175), erythrocyte-binding ligand 1 (PfEBL-1), erythrocyte-binding antigen 140 (PfEBA-140), and erythrocyte-binding antigen 181 (PfEBA-181).<ref name="Lin">PMID: 22989878</ref> PfEBA-140 binds glycophorin C on host erythrocytes,<ref name="Lin">PMID: 22989878</ref> which helps maintain erythrocyte shape and regulates membrane material properties.<ref name="Glycophorin C">http://en.wikipedia.org/wiki/Glycophorin_C</ref> Understanding the mechanism by which PfEBA-140 recognizes and engages glycophorin C on erythrocytes may lead to the future development of a new malaria vaccine, as it has been found to be immunogenic.<ref name="Ford">PMID: 17984363</ref>

==General Structure==
<Structure load='4GF2' size='400' frame='true' align='left' caption='Crystal Structure of PfEBA-140 (PDB entry [[4gf2]])' scene=''/>
[[Image:SubdomainsF1F2.png | thumb | 300px | alt=text | Subdomains of F1 and F2]]
[[Image:IndividualSubdomains.png | thumb | 300px | alt=text | Subdomains of F1 and F2]]
The EBL family members, including PfEBA-140, are made up of two regions, region II (RII) (shown to the right) and region VI.<ref name="Lin">PMID: 22989878</ref> Region II is responsible for receptor binding in all EBL family members.<ref name="Lin">PMID: 22989878</ref> RII is composed of <scene name='PfEBA-140/Rii/1'>two Duffy binding-like (DBL) domains</scene>, F1 (purple) and F2 (teal).<ref name="Lin">PMID: 22989878</ref> These two DBL domains are connected by a <scene name='PfEBA-140/Helical_linker/2'>short helical linker</scene>.<ref name="Lin">PMID: 22989878</ref> The DBL protein fold is unique to the ''Plasmodium'' species. Not only does it have the ability to recognize and bind many erythrocyte cell receptors, but it also mediates microvasculature adherence of infected erythrocytes by erythrocyte membrane protein 1 (PfEMP1).<ref name="Lin">PMID: 22989878</ref> Each DBL domain is composed of three subdomains, illustrated in the pictures to the right.

In the top image, the subdomains S1, S2, and S3 of each of the F1 and F2 domains, as well as the helical linker, are illustrated.<ref name="Lin">PMID: 22989878</ref>
In the bottom image, structures of the individual subdomains are illustrated.<ref name="Lin">PMID: 22989878</ref>

The colors are the same in both images: F1 subdomain 1 is shown in bronze, subdomain 2 in orange, subdomain 3 in dark orange; F2 subdomain 1 is shown in dark blue, subdomain 2 in blue, and subdomain 3 in light blue.

==Binding of RII PfEBA-140==
<Structure load='4gf2' size='400' frame='true' align='left' caption='Crystal Structure of PfEBA-140 (PDB entry [[4gf2]])' scene=''/>
===Requirement of Both DBL Domains===
[[Image:Binding.png | thumb | 100px | alt=text | Erythrocyte Binding of Full Length and Individual RII Domains]]Since the DBL domains of RII are highly conserved, the ability of the individual DBL domains to bind erythrocytes was tested. To do this, constructs containing the full-length RII PfEBA-140 and each individual DBL domain were tested using a rosetting assay. Both brightfield microscopy and green fluorescence protein (GFP) were used to visualize erythrocyte binding. In the upper panel, a construct expressing only GFP was used as a control. The lower panel of Figure A illustrates the extensive erythrocyte binding of the full length RII construct. In Figure B, the lack of black dots in the brightfield microscopy and the smaller quantity of green fluorescence illustrates that F1 and F2 are unable to independently bind erythrocytes. This result suggests that both domains equally participate in engaging erythrocytes.<ref name="Lin">PMID: 22989878</ref>
===Basic Patch===
[[Image:Basic Patch.png | thumb | 100px | alt=text | Erythrocyte Binding of Full Length and Individual RII Domains]]
Since the two DBL domains bind acidic GPC, there must be a number of basic charges that interact with the acidic GPC residues. This idea is supported by visualizing the <scene name='PfEBA-140/Basic_patch/1'>positive and negative residues</scene> found in RII PfEBA-140. The positive side chain residues are bue while the negative side chain residues are red. On the side shown, there are a number of residues with positive side chains, creating a basic patch. This basic patch, which forms an arch spanning the two DBL domains, is thought to interact with the acidic GPC, because if the molecule is flipped 180°, there is no concentration of positive charges. The basic patch may be better visualized in the picture to the right.<ref name="Lin">PMID: 22989878</ref>
To test whether this basic patch was indeed responsible for erythrocyte binding, two residues in each DBL domain were chosen and mutated to Alanine. In F1, these residues were <scene name='PfEBA-140/Asp247/1'>Asp247</scene> and <scene name='PfEBA-140/Arg254/1'>Arg254</scene>. In F2, these residues were <scene name='PfEBA-140/Arg485/1'>Arg485</scene> and <scene name='PfEBA-140/Asp554/2'>Asp554</scene>. When these four residues were individually mutated to Alanine, erythrocyte binding was heavily interrupted. This can be observed in the figure to the right.<ref name="Lin">PMID: 22989878</ref>[[Image:DecreasedBinding.png | thumb | 100px | alt=text | Mutation of Asp247, Arg254, Arg485, and Asp554]] The decreased erythrocyte binding confirms that these <scene name='PfEBA-140/Four/1'>four residues</scene> found in the basic patch of RII PfEBA-140 are crucial to erythrocyte binding. As a control, residues on the opposite side of RII PfEBA-140 were mutated to Alanine. These residues were Asn394 and Glu605. Mutation of these two residues had no effect on erythrocyte binding.<ref name="Lin">PMID: 22989878</ref>[[Image:FullBinding.png | thumb | 100px | alt=text | Mutation of Asn394 and Glu605]] These results fortify the finding that erythrocyte binding takes place in the basic patch discussed earlier.

 
 
 
 
 

==Structural Differences in PfEBA-140 from other EBL Ligands==
<Structure load='4gf2' size='400' frame='true' align='left' caption='Crystal Structure of PfEBA-140 (PDB entry [[4gf2]])' scene=''/>
There are three unique structural elements that set RII PfEBA-140 apart from other EBL ligands. These structures are the likely reason behind RII PfEBA-140 binding GPC so specifically.
===Disulfide Bonds===
[[Image:ModifiedPattern.png | thumb | 300px | alt=text | Disulfide Bonding Patterns]]
There are twenty-six <scene name='PfEBA-140/Cysteines/1'>cysteines</scene> in RII PfEBA-140, all of which are involved in <scene name='PfEBA-140/Disulfidebonds/1'>disulfide bonding</scene> (disulfide bonds shown in black).<ref name="Lin">PMID: 22989878</ref> In RII PfEBA-140, two modified disulfide patterns were found to be different than in RII PfEBA-175, RII PvDBP, and RII PkDBP. In the latter three RII domains, there is a disulfide linkage between Cys7 and Cys9 and between Cys8 and Cys12. In RII PfEBA-140, however, Cys7 is disulfide bonded to Cys8, and Cys9 is disulfide bonded to Cys12.<ref name="Lin">PMID: 22989878</ref>

===Alpha Helix Structure===
Another difference between RII PfEBA-140 and other EBL ligands can be found in the F2 domain. In EBL ligands of other ''Plasmodium'' species, there are β-fingers with either long or short loops which may or may not be important for receptor binding.<ref name="Lin">PMID: 22989878</ref> While this β-finger is found in F1 of PfEBA-140, it is not found in F2, as it is replaced with an <scene name='PfEBA-140/Alphabeta/1'>α-helix</scene>.<ref name="Lin">PMID: 22989878</ref>
===Lack of Kink in F1===
A third difference between RII PfEBA-140 and other EBL ligands is the lack of a glycine residue in F1. This glycine is present in F1 and F2 of PfEBA-175, PvDBP, PkDBP-α, and F2 PfEBA-140. In F2 PfEBA-140, this glycine is residue 627.<ref name="Lin">PMID: 22989878</ref> In these EBL ligands there is a kink due to glycine's flexibility. If this glycine residue were present in F1 of PfEBA-140, it would result in a kink in subdomain 3.<ref name="Lin">PMID: 22989878</ref> This kink would create a hinge angle between the two DBL domains that would allow for dimerization of PfEBA-140 upon erythrocyte binding. For this reason, PfEBA-140 is believed to engage erythrocytes as a monomer, unlike other EBL ligands, which bind erythrocytes as a dimer.
 
 
These three structural differences are believed to be responsible for the specificity of RII PfEBA-140.

==References==
<references />

Malaria Parasite Plasmodium falciparum Erythrocyte Binding Antigen 140

2012-11-24T22:31:50Z

Emily Lum: /* Binding of RII PfEBA-140 */

== Introduction ==
In 2010, malaria caused over 650,000 deaths.<ref name="World Health Organization">http://www.who.int/mediacentre/factsheets/fs094/en/index.html</ref> While this disease is both preventable and curable, many of those that live in areas where the disease is endemic do not have access to such resources. Although there is a vaccine currently undergoing clinical trials, results are not expected until 2014.<ref name="World Health Organization">http://www.who.int/mediacentre/factsheets/fs094/en/index.html</ref> The most recent vaccine research has focused on understanding a group of proteins in the erythrocyte-binding ligand (EBL) family. These proteins, which are found in the ''Plasmodium'' species, facilitate entry into erythrocytes during malarial infection by creating tight junctions between the host erythrocytes and parasite membranes. <ref name="Lin">PMID: 22989878</ref> There are four family members: erythrocyte-binding antigen 175 (PfEBA-175), erythrocyte-binding ligand 1 (PfEBL-1), erythrocyte-binding antigen 140 (PfEBA-140), and erythrocyte-binding antigen 181 (PfEBA-181).<ref name="Lin">PMID: 22989878</ref> PfEBA-140 binds glycophorin C on host erythrocytes,<ref name="Lin">PMID: 22989878</ref> which helps maintain erythrocyte shape and regulates membrane material properties.<ref name="Glycophorin C">http://en.wikipedia.org/wiki/Glycophorin_C</ref> Understanding the mechanism by which PfEBA-140 recognizes and engages glycophorin C on erythrocytes may lead to the future development of a new malaria vaccine, as it has been found to be immunogenic.<ref name="Ford">PMID: 17984363</ref>

==General Structure==
<Structure load='4GF2' size='400' frame='true' align='left' caption='Crystal Structure of PfEBA-140 (PDB entry [[4gf2]])' scene=''/>
[[Image:SubdomainsF1F2.png | thumb | 300px | alt=text | Subdomains of F1 and F2]]
[[Image:IndividualSubdomains.png | thumb | 300px | alt=text | Subdomains of F1 and F2]]
The EBL family members, including PfEBA-140, are made up of two regions, region II (RII) (shown to the right) and region VI.<ref name="Lin">PMID: 22989878</ref> Region II is responsible for receptor binding in all EBL family members.<ref name="Lin">PMID: 22989878</ref> RII is composed of <scene name='PfEBA-140/Rii/1'>two Duffy binding-like (DBL) domains</scene>, F1 (purple) and F2 (teal).<ref name="Lin">PMID: 22989878</ref> These two DBL domains are connected by a <scene name='PfEBA-140/Helical_linker/2'>short helical linker</scene>.<ref name="Lin">PMID: 22989878</ref> The DBL protein fold is unique to the ''Plasmodium'' species. Not only does it have the ability to recognize and bind many erythrocyte cell receptors, but it also mediates microvasculature adherence of infected erythrocytes by erythrocyte membrane protein 1 (PfEMP1).<ref name="Lin">PMID: 22989878</ref> Each DBL domain is composed of three subdomains, illustrated in the pictures to the right.

In the top image, the subdomains S1, S2, and S3 of each of the F1 and F2 domains, as well as the helical linker, are illustrated.<ref name="Lin">PMID: 22989878</ref>
In the bottom image, structures of the individual subdomains are illustrated.<ref name="Lin">PMID: 22989878</ref>

The colors are the same in both images: F1 subdomain 1 is shown in bronze, subdomain 2 in orange, subdomain 3 in dark orange; F2 subdomain 1 is shown in dark blue, subdomain 2 in blue, and subdomain 3 in light blue.

==Binding of RII PfEBA-140==
<Structure load='4gf2' size='400' frame='true' align='left' caption='Crystal Structure of PfEBA-140 (PDB entry [[4gf2]])' scene=''/>
===Requirement of Both DBL Domains===
[[Image:Binding.png | thumb | 100px | alt=text | Erythrocyte Binding of Full Length and Individual RII Domains]]Since the DBL domains of RII are highly conserved, the ability of the individual DBL domains to bind erythrocytes was tested. To do this, constructs containing the full-length RII PfEBA-140 and each individual DBL domain were tested using a rosetting assay. Both brightfield microscopy and green fluorescence protein (GFP) were used to visualize erythrocyte binding. In the upper panel, a construct expressing only GFP was used as a control. The lower panel of Figure A illustrates the extensive erythrocyte binding of the full length RII construct. In Figure B, the lack of black dots in the brightfield microscopy and the smaller quantity of green fluorescence illustrates that F1 and F2 are unable to independently bind erythrocytes. This result suggests that both domains equally participate in engaging erythrocytes.<ref name="Lin">PMID: 22989878</ref>
===Basic Patch===
[[Image:Basic Patch.png | thumb | 100px | alt=text | Erythrocyte Binding of Full Length and Individual RII Domains]]
Since the two DBL domains bind acidic GPC, there must be a number of basic charges that interact with the acidic GPC residues. This idea is supported by visualizing the <scene name='PfEBA-140/Basic_patch/1'>positive and negative residues</scene> found in RII PfEBA-140. The positive side chain residues are bue while the negative side chain residues are red. On the side shown, there are a number of residues with positive side chains, creating a basic patch. This basic patch, which forms an arch spanning the two DBL domains, is thought to interact with the acidic GPC, because if the molecule is flipped 180°, there is no concentration of positive charges. The basic patch may be better visualized in the picture to the right.<ref name="Lin">PMID: 22989878</ref>
To test whether this basic patch was indeed responsible for erythrocyte binding, two residues in each DBL domain were chosen and mutated to Alanine. In F1, these residues were <scene name='PfEBA-140/Asp247/1'>Asp247</scene> and <scene name='PfEBA-140/Arg254/1'>Arg254</scene>. In F2, these residues were <scene name='PfEBA-140/Arg485/1'>Arg485</scene> and <scene name='PfEBA-140/Asp554/2'>Asp554</scene>. When these four residues were individually mutated to Alanine, erythrocyte binding was heavily interrupted. This can be observed in the figure to the right.<ref name="Lin">PMID: 22989878</ref>[[Image:DecreasedBinding.png | thumb | 100px | alt=text | Mutation of Asp247, Arg254, Arg485, and Asp554]] The decreased erythrocyte binding confirms that these <scene name='PfEBA-140/Four/1'>four residues</scene> found in the basic patch of RII PfEBA-140 are crucial to erythrocyte binding. As a control, residues on the opposite side of RII PfEBA-140 were mutated to Alanine. These residues were Asn394 and Glu605. Mutation of these two residues had no effect on erythrocyte binding.<ref name="Lin">PMID: 22989878</ref>[[Image:FullBinding.png | thumb | 100px | alt=text | Mutation of Asn394 and Glu605]] These results fortify the finding that erythrocyte binding takes place in the basic patch discussed earlier.

 
 
 
 
 

==Structural Differences in PfEBA-140 from other EBL Ligands==
<Structure load='4gf2' size='400' frame='true' align='left' caption='Crystal Structure of PfEBA-140 (PDB entry [[4GF2]])' scene=''/>
There are three unique structural elements that set RII PfEBA-140 apart from other EBL ligands. These structures are the likely reason behind RII PfEBA-140 binding GPC so specifically.
===Disulfide Bonds===
[[Image:ModifiedPattern.png | thumb | 300px | alt=text | Disulfide Bonding Patterns]]
There are twenty-six <scene name='PfEBA-140/Cysteines/1'>cysteines</scene> in RII PfEBA-140, all of which are involved in <scene name='PfEBA-140/Disulfidebonds/1'>disulfide bonding</scene> (disulfide bonds shown in black).<ref name="Lin">PMID: 22989878</ref> In RII PfEBA-140, two modified disulfide patterns were found to be different than in RII PfEBA-175, RII PvDBP, and RII PkDBP. In the latter three RII domains, there is a disulfide linkage between Cys7 and Cys9 and between Cys8 and Cys12. In RII PfEBA-140, however, Cys7 is disulfide bonded to Cys8, and Cys9 is disulfide bonded to Cys12.<ref name="Lin">PMID: 22989878</ref>

===Alpha Helix Structure===
Another difference between RII PfEBA-140 and other EBL ligands can be found in the F2 domain. In EBL ligands of other ''Plasmodium'' species, there are β-fingers with either long or short loops which may or may not be important for receptor binding.<ref name="Lin">PMID: 22989878</ref> While this β-finger is found in F1 of PfEBA-140, it is not found in F2, as it is replaced with an <scene name='PfEBA-140/Alphabeta/1'>α-helix</scene>.<ref name="Lin">PMID: 22989878</ref>
===Lack of Kink in F1===
A third difference between RII PfEBA-140 and other EBL ligands is the lack of a glycine residue in F1. This glycine is present in F1 and F2 of PfEBA-175, PvDBP, PkDBP-α, and F2 PfEBA-140. In F2 PfEBA-140, this glycine is residue 627.<ref name="Lin">PMID: 22989878</ref> In these EBL ligands there is a kink due to glycine's flexibility. If this glycine residue were present in F1 of PfEBA-140, it would result in a kink in subdomain 3.<ref name="Lin">PMID: 22989878</ref> This kink would create a hinge angle between the two DBL domains that would allow for dimerization of PfEBA-140 upon erythrocyte binding. For this reason, PfEBA-140 is believed to engage erythrocytes as a monomer, unlike other EBL ligands, which bind erythrocytes as a dimer.
 
 
These three structural differences are believed to be responsible for the specificity of RII PfEBA-140.

==References==
<references />

Malaria Parasite Plasmodium falciparum Erythrocyte Binding Antigen 140

2012-11-24T22:25:46Z

Emily Lum: /* Structural Differences in PfEBA-140 from other EBL Ligands */

== Introduction ==
In 2010, malaria caused over 650,000 deaths.<ref name="World Health Organization">http://www.who.int/mediacentre/factsheets/fs094/en/index.html</ref> While this disease is both preventable and curable, many of those that live in areas where the disease is endemic do not have access to such resources. Although there is a vaccine currently undergoing clinical trials, results are not expected until 2014.<ref name="World Health Organization">http://www.who.int/mediacentre/factsheets/fs094/en/index.html</ref> The most recent vaccine research has focused on understanding a group of proteins in the erythrocyte-binding ligand (EBL) family. These proteins, which are found in the ''Plasmodium'' species, facilitate entry into erythrocytes during malarial infection by creating tight junctions between the host erythrocytes and parasite membranes. <ref name="Lin">PMID: 22989878</ref> There are four family members: erythrocyte-binding antigen 175 (PfEBA-175), erythrocyte-binding ligand 1 (PfEBL-1), erythrocyte-binding antigen 140 (PfEBA-140), and erythrocyte-binding antigen 181 (PfEBA-181).<ref name="Lin">PMID: 22989878</ref> PfEBA-140 binds glycophorin C on host erythrocytes,<ref name="Lin">PMID: 22989878</ref> which helps maintain erythrocyte shape and regulates membrane material properties.<ref name="Glycophorin C">http://en.wikipedia.org/wiki/Glycophorin_C</ref> Understanding the mechanism by which PfEBA-140 recognizes and engages glycophorin C on erythrocytes may lead to the future development of a new malaria vaccine, as it has been found to be immunogenic.<ref name="Ford">PMID: 17984363</ref>

==General Structure==
<Structure load='4GF2' size='400' frame='true' align='left' caption='Crystal Structure of PfEBA-140 (PDB entry [[4gf2]])' scene=''/>
[[Image:SubdomainsF1F2.png | thumb | 300px | alt=text | Subdomains of F1 and F2]]
[[Image:IndividualSubdomains.png | thumb | 300px | alt=text | Subdomains of F1 and F2]]
The EBL family members, including PfEBA-140, are made up of two regions, region II (RII) (shown to the right) and region VI.<ref name="Lin">PMID: 22989878</ref> Region II is responsible for receptor binding in all EBL family members.<ref name="Lin">PMID: 22989878</ref> RII is composed of <scene name='PfEBA-140/Rii/1'>two Duffy binding-like (DBL) domains</scene>, F1 (purple) and F2 (teal).<ref name="Lin">PMID: 22989878</ref> These two DBL domains are connected by a <scene name='PfEBA-140/Helical_linker/2'>short helical linker</scene>.<ref name="Lin">PMID: 22989878</ref> The DBL protein fold is unique to the ''Plasmodium'' species. Not only does it have the ability to recognize and bind many erythrocyte cell receptors, but it also mediates microvasculature adherence of infected erythrocytes by erythrocyte membrane protein 1 (PfEMP1).<ref name="Lin">PMID: 22989878</ref> Each DBL domain is composed of three subdomains, illustrated in the pictures to the right.

In the top image, the subdomains S1, S2, and S3 of each of the F1 and F2 domains, as well as the helical linker, are illustrated.<ref name="Lin">PMID: 22989878</ref>
In the bottom image, structures of the individual subdomains are illustrated.<ref name="Lin">PMID: 22989878</ref>

The colors are the same in both images: F1 subdomain 1 is shown in bronze, subdomain 2 in orange, subdomain 3 in dark orange; F2 subdomain 1 is shown in dark blue, subdomain 2 in blue, and subdomain 3 in light blue.

==Binding of RII PfEBA-140==
<Structure load='4gf2' size='400' frame='true' align='left' caption='Crystal Structure of PfEBA-140 (PDB entry [[4GF2]])' scene=''/>
===Requirement of Both DBL Domains===
[[Image:Binding.png | thumb | 100px | alt=text | Erythrocyte Binding of Full Length and Individual RII Domains]]Since the DBL domains of RII are highly conserved, the ability of the individual DBL domains to bind erythrocytes was tested. To do this, constructs containing the full-length RII PfEBA-140 and each individual DBL domain were tested using a rosetting assay. Both brightfield microscopy and green fluorescence protein (GFP) were used to visualize erythrocyte binding. In the upper panel, a construct expressing only GFP was used as a control. The lower panel of Figure A illustrates the extensive erythrocyte binding of the full length RII construct. In Figure B, the lack of black dots in the brightfield microscopy and the smaller quantity of green fluorescence illustrates that F1 and F2 are unable to independently bind erythrocytes. This result suggests that both domains equally participate in engaging erythrocytes.<ref name="Lin">PMID: 22989878</ref>
===Basic Patch===
[[Image:Basic Patch.png | thumb | 100px | alt=text | Erythrocyte Binding of Full Length and Individual RII Domains]]
Since the two DBL domains bind acidic GPC, there must be a number of basic charges that interact with the acidic GPC residues. This idea is supported by visualizing the <scene name='PfEBA-140/Basic_patch/1'>positive and negative residues</scene> found in RII PfEBA-140. The positive side chain residues are bue while the negative side chain residues are red. On the side shown, there are a number of residues with positive side chains, creating a basic patch. This basic patch, which forms an arch spanning the two DBL domains, is thought to interact with the acidic GPC, because if the molecule is flipped 180°, there is no concentration of positive charges. The basic patch may be better visualized in the picture to the right.<ref name="Lin">PMID: 22989878</ref>
To test whether this basic patch was indeed responsible for erythrocyte binding, two residues in each DBL domain were chosen and mutated to Alanine. In F1, these residues were <scene name='PfEBA-140/Asp247/1'>Asp247</scene> and <scene name='PfEBA-140/Arg254/1'>Arg254</scene>. In F2, these residues were <scene name='PfEBA-140/Arg485/1'>Arg485</scene> and <scene name='PfEBA-140/Asp554/2'>Asp554</scene>. When these four residues were individually mutated to Alanine, erythrocyte binding was heavily interrupted. This can be observed in the figure to the right.<ref name="Lin">PMID: 22989878</ref>[[Image:DecreasedBinding.png | thumb | 100px | alt=text | Mutation of Asp247, Arg254, Arg485, and Asp554]] The decreased erythrocyte binding confirms that these <scene name='PfEBA-140/Four/1'>four residues</scene> found in the basic patch of RII PfEBA-140 are crucial to erythrocyte binding. As a control, residues on the opposite side of RII PfEBA-140 were mutated to Alanine. These residues were Asn394 and Glu605. Mutation of these two residues had no effect on erythrocyte binding.<ref name="Lin">PMID: 22989878</ref>[[Image:FullBinding.png | thumb | 100px | alt=text | Mutation of Asn394 and Glu605]] These results fortify the finding that erythrocyte binding takes place in the basic patch discussed earlier.

 
 
 
 
 

==Structural Differences in PfEBA-140 from other EBL Ligands==
<Structure load='4gf2' size='400' frame='true' align='left' caption='Crystal Structure of PfEBA-140 (PDB entry [[4GF2]])' scene=''/>
There are three unique structural elements that set RII PfEBA-140 apart from other EBL ligands. These structures are the likely reason behind RII PfEBA-140 binding GPC so specifically.
===Disulfide Bonds===
[[Image:ModifiedPattern.png | thumb | 300px | alt=text | Disulfide Bonding Patterns]]
There are twenty-six <scene name='PfEBA-140/Cysteines/1'>cysteines</scene> in RII PfEBA-140, all of which are involved in <scene name='PfEBA-140/Disulfidebonds/1'>disulfide bonding</scene> (disulfide bonds shown in black).<ref name="Lin">PMID: 22989878</ref> In RII PfEBA-140, two modified disulfide patterns were found to be different than in RII PfEBA-175, RII PvDBP, and RII PkDBP. In the latter three RII domains, there is a disulfide linkage between Cys7 and Cys9 and between Cys8 and Cys12. In RII PfEBA-140, however, Cys7 is disulfide bonded to Cys8, and Cys9 is disulfide bonded to Cys12.<ref name="Lin">PMID: 22989878</ref>

===Alpha Helix Structure===
Another difference between RII PfEBA-140 and other EBL ligands can be found in the F2 domain. In EBL ligands of other ''Plasmodium'' species, there are β-fingers with either long or short loops which may or may not be important for receptor binding.<ref name="Lin">PMID: 22989878</ref> While this β-finger is found in F1 of PfEBA-140, it is not found in F2, as it is replaced with an <scene name='PfEBA-140/Alphabeta/1'>α-helix</scene>.<ref name="Lin">PMID: 22989878</ref>
===Lack of Kink in F1===
A third difference between RII PfEBA-140 and other EBL ligands is the lack of a glycine residue in F1. This glycine is present in F1 and F2 of PfEBA-175, PvDBP, PkDBP-α, and F2 PfEBA-140. In F2 PfEBA-140, this glycine is residue 627.<ref name="Lin">PMID: 22989878</ref> In these EBL ligands there is a kink due to glycine's flexibility. If this glycine residue were present in F1 of PfEBA-140, it would result in a kink in subdomain 3.<ref name="Lin">PMID: 22989878</ref> This kink would create a hinge angle between the two DBL domains that would allow for dimerization of PfEBA-140 upon erythrocyte binding. For this reason, PfEBA-140 is believed to engage erythrocytes as a monomer, unlike other EBL ligands, which bind erythrocytes as a dimer.
 
 
These three structural differences are believed to be responsible for the specificity of RII PfEBA-140.

==References==
<references />

Malaria Parasite Plasmodium falciparum Erythrocyte Binding Antigen 140

2012-11-24T22:25:26Z

Emily Lum: /* Binding of RII PfEBA-140 */

== Introduction ==
In 2010, malaria caused over 650,000 deaths.<ref name="World Health Organization">http://www.who.int/mediacentre/factsheets/fs094/en/index.html</ref> While this disease is both preventable and curable, many of those that live in areas where the disease is endemic do not have access to such resources. Although there is a vaccine currently undergoing clinical trials, results are not expected until 2014.<ref name="World Health Organization">http://www.who.int/mediacentre/factsheets/fs094/en/index.html</ref> The most recent vaccine research has focused on understanding a group of proteins in the erythrocyte-binding ligand (EBL) family. These proteins, which are found in the ''Plasmodium'' species, facilitate entry into erythrocytes during malarial infection by creating tight junctions between the host erythrocytes and parasite membranes. <ref name="Lin">PMID: 22989878</ref> There are four family members: erythrocyte-binding antigen 175 (PfEBA-175), erythrocyte-binding ligand 1 (PfEBL-1), erythrocyte-binding antigen 140 (PfEBA-140), and erythrocyte-binding antigen 181 (PfEBA-181).<ref name="Lin">PMID: 22989878</ref> PfEBA-140 binds glycophorin C on host erythrocytes,<ref name="Lin">PMID: 22989878</ref> which helps maintain erythrocyte shape and regulates membrane material properties.<ref name="Glycophorin C">http://en.wikipedia.org/wiki/Glycophorin_C</ref> Understanding the mechanism by which PfEBA-140 recognizes and engages glycophorin C on erythrocytes may lead to the future development of a new malaria vaccine, as it has been found to be immunogenic.<ref name="Ford">PMID: 17984363</ref>

==General Structure==
<Structure load='4GF2' size='400' frame='true' align='left' caption='Crystal Structure of PfEBA-140 (PDB entry [[4gf2]])' scene=''/>
[[Image:SubdomainsF1F2.png | thumb | 300px | alt=text | Subdomains of F1 and F2]]
[[Image:IndividualSubdomains.png | thumb | 300px | alt=text | Subdomains of F1 and F2]]
The EBL family members, including PfEBA-140, are made up of two regions, region II (RII) (shown to the right) and region VI.<ref name="Lin">PMID: 22989878</ref> Region II is responsible for receptor binding in all EBL family members.<ref name="Lin">PMID: 22989878</ref> RII is composed of <scene name='PfEBA-140/Rii/1'>two Duffy binding-like (DBL) domains</scene>, F1 (purple) and F2 (teal).<ref name="Lin">PMID: 22989878</ref> These two DBL domains are connected by a <scene name='PfEBA-140/Helical_linker/2'>short helical linker</scene>.<ref name="Lin">PMID: 22989878</ref> The DBL protein fold is unique to the ''Plasmodium'' species. Not only does it have the ability to recognize and bind many erythrocyte cell receptors, but it also mediates microvasculature adherence of infected erythrocytes by erythrocyte membrane protein 1 (PfEMP1).<ref name="Lin">PMID: 22989878</ref> Each DBL domain is composed of three subdomains, illustrated in the pictures to the right.

In the top image, the subdomains S1, S2, and S3 of each of the F1 and F2 domains, as well as the helical linker, are illustrated.<ref name="Lin">PMID: 22989878</ref>
In the bottom image, structures of the individual subdomains are illustrated.<ref name="Lin">PMID: 22989878</ref>

The colors are the same in both images: F1 subdomain 1 is shown in bronze, subdomain 2 in orange, subdomain 3 in dark orange; F2 subdomain 1 is shown in dark blue, subdomain 2 in blue, and subdomain 3 in light blue.

==Binding of RII PfEBA-140==
<Structure load='4gf2' size='400' frame='true' align='left' caption='Crystal Structure of PfEBA-140 (PDB entry [[4GF2]])' scene=''/>
===Requirement of Both DBL Domains===
[[Image:Binding.png | thumb | 100px | alt=text | Erythrocyte Binding of Full Length and Individual RII Domains]]Since the DBL domains of RII are highly conserved, the ability of the individual DBL domains to bind erythrocytes was tested. To do this, constructs containing the full-length RII PfEBA-140 and each individual DBL domain were tested using a rosetting assay. Both brightfield microscopy and green fluorescence protein (GFP) were used to visualize erythrocyte binding. In the upper panel, a construct expressing only GFP was used as a control. The lower panel of Figure A illustrates the extensive erythrocyte binding of the full length RII construct. In Figure B, the lack of black dots in the brightfield microscopy and the smaller quantity of green fluorescence illustrates that F1 and F2 are unable to independently bind erythrocytes. This result suggests that both domains equally participate in engaging erythrocytes.<ref name="Lin">PMID: 22989878</ref>
===Basic Patch===
[[Image:Basic Patch.png | thumb | 100px | alt=text | Erythrocyte Binding of Full Length and Individual RII Domains]]
Since the two DBL domains bind acidic GPC, there must be a number of basic charges that interact with the acidic GPC residues. This idea is supported by visualizing the <scene name='PfEBA-140/Basic_patch/1'>positive and negative residues</scene> found in RII PfEBA-140. The positive side chain residues are bue while the negative side chain residues are red. On the side shown, there are a number of residues with positive side chains, creating a basic patch. This basic patch, which forms an arch spanning the two DBL domains, is thought to interact with the acidic GPC, because if the molecule is flipped 180°, there is no concentration of positive charges. The basic patch may be better visualized in the picture to the right.<ref name="Lin">PMID: 22989878</ref>
To test whether this basic patch was indeed responsible for erythrocyte binding, two residues in each DBL domain were chosen and mutated to Alanine. In F1, these residues were <scene name='PfEBA-140/Asp247/1'>Asp247</scene> and <scene name='PfEBA-140/Arg254/1'>Arg254</scene>. In F2, these residues were <scene name='PfEBA-140/Arg485/1'>Arg485</scene> and <scene name='PfEBA-140/Asp554/2'>Asp554</scene>. When these four residues were individually mutated to Alanine, erythrocyte binding was heavily interrupted. This can be observed in the figure to the right.<ref name="Lin">PMID: 22989878</ref>[[Image:DecreasedBinding.png | thumb | 100px | alt=text | Mutation of Asp247, Arg254, Arg485, and Asp554]] The decreased erythrocyte binding confirms that these <scene name='PfEBA-140/Four/1'>four residues</scene> found in the basic patch of RII PfEBA-140 are crucial to erythrocyte binding. As a control, residues on the opposite side of RII PfEBA-140 were mutated to Alanine. These residues were Asn394 and Glu605. Mutation of these two residues had no effect on erythrocyte binding.<ref name="Lin">PMID: 22989878</ref>[[Image:FullBinding.png | thumb | 100px | alt=text | Mutation of Asn394 and Glu605]] These results fortify the finding that erythrocyte binding takes place in the basic patch discussed earlier.

 
 
 
 
 

==Structural Differences in PfEBA-140 from other EBL Ligands==
<Structure load='4GF2' size='400' frame='true' align='left' caption='Crystal Structure of PfEBA-140 (PDB entry [[4GF2]])' scene=''/>
There are three unique structural elements that set RII PfEBA-140 apart from other EBL ligands. These structures are the likely reason behind RII PfEBA-140 binding GPC so specifically.
===Disulfide Bonds===
[[Image:ModifiedPattern.png | thumb | 300px | alt=text | Disulfide Bonding Patterns]]
There are twenty-six <scene name='PfEBA-140/Cysteines/1'>cysteines</scene> in RII PfEBA-140, all of which are involved in <scene name='PfEBA-140/Disulfidebonds/1'>disulfide bonding</scene> (disulfide bonds shown in black).<ref name="Lin">PMID: 22989878</ref> In RII PfEBA-140, two modified disulfide patterns were found to be different than in RII PfEBA-175, RII PvDBP, and RII PkDBP. In the latter three RII domains, there is a disulfide linkage between Cys7 and Cys9 and between Cys8 and Cys12. In RII PfEBA-140, however, Cys7 is disulfide bonded to Cys8, and Cys9 is disulfide bonded to Cys12.<ref name="Lin">PMID: 22989878</ref>

===Alpha Helix Structure===
Another difference between RII PfEBA-140 and other EBL ligands can be found in the F2 domain. In EBL ligands of other ''Plasmodium'' species, there are β-fingers with either long or short loops which may or may not be important for receptor binding.<ref name="Lin">PMID: 22989878</ref> While this β-finger is found in F1 of PfEBA-140, it is not found in F2, as it is replaced with an <scene name='PfEBA-140/Alphabeta/1'>α-helix</scene>.<ref name="Lin">PMID: 22989878</ref>
===Lack of Kink in F1===
A third difference between RII PfEBA-140 and other EBL ligands is the lack of a glycine residue in F1. This glycine is present in F1 and F2 of PfEBA-175, PvDBP, PkDBP-α, and F2 PfEBA-140. In F2 PfEBA-140, this glycine is residue 627.<ref name="Lin">PMID: 22989878</ref> In these EBL ligands there is a kink due to glycine's flexibility. If this glycine residue were present in F1 of PfEBA-140, it would result in a kink in subdomain 3.<ref name="Lin">PMID: 22989878</ref> This kink would create a hinge angle between the two DBL domains that would allow for dimerization of PfEBA-140 upon erythrocyte binding. For this reason, PfEBA-140 is believed to engage erythrocytes as a monomer, unlike other EBL ligands, which bind erythrocytes as a dimer.
 
 
These three structural differences are believed to be responsible for the specificity of RII PfEBA-140.

==References==
<references />

Malaria Parasite Plasmodium falciparum Erythrocyte Binding Antigen 140

2012-11-24T22:24:32Z

Emily Lum: /* General Structure */

== Introduction ==
In 2010, malaria caused over 650,000 deaths.<ref name="World Health Organization">http://www.who.int/mediacentre/factsheets/fs094/en/index.html</ref> While this disease is both preventable and curable, many of those that live in areas where the disease is endemic do not have access to such resources. Although there is a vaccine currently undergoing clinical trials, results are not expected until 2014.<ref name="World Health Organization">http://www.who.int/mediacentre/factsheets/fs094/en/index.html</ref> The most recent vaccine research has focused on understanding a group of proteins in the erythrocyte-binding ligand (EBL) family. These proteins, which are found in the ''Plasmodium'' species, facilitate entry into erythrocytes during malarial infection by creating tight junctions between the host erythrocytes and parasite membranes. <ref name="Lin">PMID: 22989878</ref> There are four family members: erythrocyte-binding antigen 175 (PfEBA-175), erythrocyte-binding ligand 1 (PfEBL-1), erythrocyte-binding antigen 140 (PfEBA-140), and erythrocyte-binding antigen 181 (PfEBA-181).<ref name="Lin">PMID: 22989878</ref> PfEBA-140 binds glycophorin C on host erythrocytes,<ref name="Lin">PMID: 22989878</ref> which helps maintain erythrocyte shape and regulates membrane material properties.<ref name="Glycophorin C">http://en.wikipedia.org/wiki/Glycophorin_C</ref> Understanding the mechanism by which PfEBA-140 recognizes and engages glycophorin C on erythrocytes may lead to the future development of a new malaria vaccine, as it has been found to be immunogenic.<ref name="Ford">PMID: 17984363</ref>

==General Structure==
<Structure load='4GF2' size='400' frame='true' align='left' caption='Crystal Structure of PfEBA-140 (PDB entry [[4gf2]])' scene=''/>
[[Image:SubdomainsF1F2.png | thumb | 300px | alt=text | Subdomains of F1 and F2]]
[[Image:IndividualSubdomains.png | thumb | 300px | alt=text | Subdomains of F1 and F2]]
The EBL family members, including PfEBA-140, are made up of two regions, region II (RII) (shown to the right) and region VI.<ref name="Lin">PMID: 22989878</ref> Region II is responsible for receptor binding in all EBL family members.<ref name="Lin">PMID: 22989878</ref> RII is composed of <scene name='PfEBA-140/Rii/1'>two Duffy binding-like (DBL) domains</scene>, F1 (purple) and F2 (teal).<ref name="Lin">PMID: 22989878</ref> These two DBL domains are connected by a <scene name='PfEBA-140/Helical_linker/2'>short helical linker</scene>.<ref name="Lin">PMID: 22989878</ref> The DBL protein fold is unique to the ''Plasmodium'' species. Not only does it have the ability to recognize and bind many erythrocyte cell receptors, but it also mediates microvasculature adherence of infected erythrocytes by erythrocyte membrane protein 1 (PfEMP1).<ref name="Lin">PMID: 22989878</ref> Each DBL domain is composed of three subdomains, illustrated in the pictures to the right.

In the top image, the subdomains S1, S2, and S3 of each of the F1 and F2 domains, as well as the helical linker, are illustrated.<ref name="Lin">PMID: 22989878</ref>
In the bottom image, structures of the individual subdomains are illustrated.<ref name="Lin">PMID: 22989878</ref>

The colors are the same in both images: F1 subdomain 1 is shown in bronze, subdomain 2 in orange, subdomain 3 in dark orange; F2 subdomain 1 is shown in dark blue, subdomain 2 in blue, and subdomain 3 in light blue.

==Binding of RII PfEBA-140==
<Structure load='4GF2' size='400' frame='true' align='left' caption='Crystal Structure of PfEBA-140 (PDB entry [[4GF2]])' scene=''/>
===Requirement of Both DBL Domains===
[[Image:Binding.png | thumb | 100px | alt=text | Erythrocyte Binding of Full Length and Individual RII Domains]]Since the DBL domains of RII are highly conserved, the ability of the individual DBL domains to bind erythrocytes was tested. To do this, constructs containing the full-length RII PfEBA-140 and each individual DBL domain were tested using a rosetting assay. Both brightfield microscopy and green fluorescence protein (GFP) were used to visualize erythrocyte binding. In the upper panel, a construct expressing only GFP was used as a control. The lower panel of Figure A illustrates the extensive erythrocyte binding of the full length RII construct. In Figure B, the lack of black dots in the brightfield microscopy and the smaller quantity of green fluorescence illustrates that F1 and F2 are unable to independently bind erythrocytes. This result suggests that both domains equally participate in engaging erythrocytes.<ref name="Lin">PMID: 22989878</ref>
===Basic Patch===
[[Image:Basic Patch.png | thumb | 100px | alt=text | Erythrocyte Binding of Full Length and Individual RII Domains]]
Since the two DBL domains bind acidic GPC, there must be a number of basic charges that interact with the acidic GPC residues. This idea is supported by visualizing the <scene name='PfEBA-140/Basic_patch/1'>positive and negative residues</scene> found in RII PfEBA-140. The positive side chain residues are bue while the negative side chain residues are red. On the side shown, there are a number of residues with positive side chains, creating a basic patch. This basic patch, which forms an arch spanning the two DBL domains, is thought to interact with the acidic GPC, because if the molecule is flipped 180°, there is no concentration of positive charges. The basic patch may be better visualized in the picture to the right.<ref name="Lin">PMID: 22989878</ref>
To test whether this basic patch was indeed responsible for erythrocyte binding, two residues in each DBL domain were chosen and mutated to Alanine. In F1, these residues were <scene name='PfEBA-140/Asp247/1'>Asp247</scene> and <scene name='PfEBA-140/Arg254/1'>Arg254</scene>. In F2, these residues were <scene name='PfEBA-140/Arg485/1'>Arg485</scene> and <scene name='PfEBA-140/Asp554/2'>Asp554</scene>. When these four residues were individually mutated to Alanine, erythrocyte binding was heavily interrupted. This can be observed in the figure to the right.<ref name="Lin">PMID: 22989878</ref>[[Image:DecreasedBinding.png | thumb | 100px | alt=text | Mutation of Asp247, Arg254, Arg485, and Asp554]] The decreased erythrocyte binding confirms that these <scene name='PfEBA-140/Four/1'>four residues</scene> found in the basic patch of RII PfEBA-140 are crucial to erythrocyte binding. As a control, residues on the opposite side of RII PfEBA-140 were mutated to Alanine. These residues were Asn394 and Glu605. Mutation of these two residues had no effect on erythrocyte binding.<ref name="Lin">PMID: 22989878</ref>[[Image:FullBinding.png | thumb | 100px | alt=text | Mutation of Asn394 and Glu605]] These results fortify the finding that erythrocyte binding takes place in the basic patch discussed earlier.

 
 
 
 
 

==Structural Differences in PfEBA-140 from other EBL Ligands==
<Structure load='4GF2' size='400' frame='true' align='left' caption='Crystal Structure of PfEBA-140 (PDB entry [[4GF2]])' scene=''/>
There are three unique structural elements that set RII PfEBA-140 apart from other EBL ligands. These structures are the likely reason behind RII PfEBA-140 binding GPC so specifically.
===Disulfide Bonds===
[[Image:ModifiedPattern.png | thumb | 300px | alt=text | Disulfide Bonding Patterns]]
There are twenty-six <scene name='PfEBA-140/Cysteines/1'>cysteines</scene> in RII PfEBA-140, all of which are involved in <scene name='PfEBA-140/Disulfidebonds/1'>disulfide bonding</scene> (disulfide bonds shown in black).<ref name="Lin">PMID: 22989878</ref> In RII PfEBA-140, two modified disulfide patterns were found to be different than in RII PfEBA-175, RII PvDBP, and RII PkDBP. In the latter three RII domains, there is a disulfide linkage between Cys7 and Cys9 and between Cys8 and Cys12. In RII PfEBA-140, however, Cys7 is disulfide bonded to Cys8, and Cys9 is disulfide bonded to Cys12.<ref name="Lin">PMID: 22989878</ref>

===Alpha Helix Structure===
Another difference between RII PfEBA-140 and other EBL ligands can be found in the F2 domain. In EBL ligands of other ''Plasmodium'' species, there are β-fingers with either long or short loops which may or may not be important for receptor binding.<ref name="Lin">PMID: 22989878</ref> While this β-finger is found in F1 of PfEBA-140, it is not found in F2, as it is replaced with an <scene name='PfEBA-140/Alphabeta/1'>α-helix</scene>.<ref name="Lin">PMID: 22989878</ref>
===Lack of Kink in F1===
A third difference between RII PfEBA-140 and other EBL ligands is the lack of a glycine residue in F1. This glycine is present in F1 and F2 of PfEBA-175, PvDBP, PkDBP-α, and F2 PfEBA-140. In F2 PfEBA-140, this glycine is residue 627.<ref name="Lin">PMID: 22989878</ref> In these EBL ligands there is a kink due to glycine's flexibility. If this glycine residue were present in F1 of PfEBA-140, it would result in a kink in subdomain 3.<ref name="Lin">PMID: 22989878</ref> This kink would create a hinge angle between the two DBL domains that would allow for dimerization of PfEBA-140 upon erythrocyte binding. For this reason, PfEBA-140 is believed to engage erythrocytes as a monomer, unlike other EBL ligands, which bind erythrocytes as a dimer.
 
 
These three structural differences are believed to be responsible for the specificity of RII PfEBA-140.

==References==
<references />

User:Emily Lum/PfEBA-140

2012-11-24T22:23:12Z

Emily Lum: User:Emily Lum/PfEBA-140 moved to Malaria Parasite Plasmodium falciparum Erythrocyte Binding Antigen 140: no longer need page to only be edited by myself

#REDIRECT [[Malaria Parasite Plasmodium falciparum Erythrocyte Binding Antigen 140]]

Malaria Parasite Plasmodium falciparum Erythrocyte Binding Antigen 140

2012-11-24T22:23:12Z

Emily Lum: User:Emily Lum/PfEBA-140 moved to Malaria Parasite Plasmodium falciparum Erythrocyte Binding Antigen 140: no longer need page to only be edited by myself

== Introduction ==
In 2010, malaria caused over 650,000 deaths.<ref name="World Health Organization">http://www.who.int/mediacentre/factsheets/fs094/en/index.html</ref> While this disease is both preventable and curable, many of those that live in areas where the disease is endemic do not have access to such resources. Although there is a vaccine currently undergoing clinical trials, results are not expected until 2014.<ref name="World Health Organization">http://www.who.int/mediacentre/factsheets/fs094/en/index.html</ref> The most recent vaccine research has focused on understanding a group of proteins in the erythrocyte-binding ligand (EBL) family. These proteins, which are found in the ''Plasmodium'' species, facilitate entry into erythrocytes during malarial infection by creating tight junctions between the host erythrocytes and parasite membranes. <ref name="Lin">PMID: 22989878</ref> There are four family members: erythrocyte-binding antigen 175 (PfEBA-175), erythrocyte-binding ligand 1 (PfEBL-1), erythrocyte-binding antigen 140 (PfEBA-140), and erythrocyte-binding antigen 181 (PfEBA-181).<ref name="Lin">PMID: 22989878</ref> PfEBA-140 binds glycophorin C on host erythrocytes,<ref name="Lin">PMID: 22989878</ref> which helps maintain erythrocyte shape and regulates membrane material properties.<ref name="Glycophorin C">http://en.wikipedia.org/wiki/Glycophorin_C</ref> Understanding the mechanism by which PfEBA-140 recognizes and engages glycophorin C on erythrocytes may lead to the future development of a new malaria vaccine, as it has been found to be immunogenic.<ref name="Ford">PMID: 17984363</ref>

==General Structure==
<Structure load='4GF2' size='400' frame='true' align='left' caption='Crystal Structure of PfEBA-140 (PDB entry [[4GF2]])' scene=''/>
[[Image:SubdomainsF1F2.png | thumb | 300px | alt=text | Subdomains of F1 and F2]]
[[Image:IndividualSubdomains.png | thumb | 300px | alt=text | Subdomains of F1 and F2]]
The EBL family members, including PfEBA-140, are made up of two regions, region II (RII) (shown to the right) and region VI.<ref name="Lin">PMID: 22989878</ref> Region II is responsible for receptor binding in all EBL family members.<ref name="Lin">PMID: 22989878</ref> RII is composed of <scene name='PfEBA-140/Rii/1'>two Duffy binding-like (DBL) domains</scene>, F1 (purple) and F2 (teal).<ref name="Lin">PMID: 22989878</ref> These two DBL domains are connected by a <scene name='PfEBA-140/Helical_linker/2'>short helical linker</scene>.<ref name="Lin">PMID: 22989878</ref> The DBL protein fold is unique to the ''Plasmodium'' species. Not only does it have the ability to recognize and bind many erythrocyte cell receptors, but it also mediates microvasculature adherence of infected erythrocytes by erythrocyte membrane protein 1 (PfEMP1).<ref name="Lin">PMID: 22989878</ref> Each DBL domain is composed of three subdomains, illustrated in the pictures to the right.

In the top image, the subdomains S1, S2, and S3 of each of the F1 and F2 domains, as well as the helical linker, are illustrated.<ref name="Lin">PMID: 22989878</ref>
In the bottom image, structures of the individual subdomains are illustrated.<ref name="Lin">PMID: 22989878</ref>

The colors are the same in both images: F1 subdomain 1 is shown in bronze, subdomain 2 in orange, subdomain 3 in dark orange; F2 subdomain 1 is shown in dark blue, subdomain 2 in blue, and subdomain 3 in light blue.

==Binding of RII PfEBA-140==
<Structure load='4GF2' size='400' frame='true' align='left' caption='Crystal Structure of PfEBA-140 (PDB entry [[4GF2]])' scene=''/>
===Requirement of Both DBL Domains===
[[Image:Binding.png | thumb | 100px | alt=text | Erythrocyte Binding of Full Length and Individual RII Domains]]Since the DBL domains of RII are highly conserved, the ability of the individual DBL domains to bind erythrocytes was tested. To do this, constructs containing the full-length RII PfEBA-140 and each individual DBL domain were tested using a rosetting assay. Both brightfield microscopy and green fluorescence protein (GFP) were used to visualize erythrocyte binding. In the upper panel, a construct expressing only GFP was used as a control. The lower panel of Figure A illustrates the extensive erythrocyte binding of the full length RII construct. In Figure B, the lack of black dots in the brightfield microscopy and the smaller quantity of green fluorescence illustrates that F1 and F2 are unable to independently bind erythrocytes. This result suggests that both domains equally participate in engaging erythrocytes.<ref name="Lin">PMID: 22989878</ref>
===Basic Patch===
[[Image:Basic Patch.png | thumb | 100px | alt=text | Erythrocyte Binding of Full Length and Individual RII Domains]]
Since the two DBL domains bind acidic GPC, there must be a number of basic charges that interact with the acidic GPC residues. This idea is supported by visualizing the <scene name='PfEBA-140/Basic_patch/1'>positive and negative residues</scene> found in RII PfEBA-140. The positive side chain residues are bue while the negative side chain residues are red. On the side shown, there are a number of residues with positive side chains, creating a basic patch. This basic patch, which forms an arch spanning the two DBL domains, is thought to interact with the acidic GPC, because if the molecule is flipped 180°, there is no concentration of positive charges. The basic patch may be better visualized in the picture to the right.<ref name="Lin">PMID: 22989878</ref>
To test whether this basic patch was indeed responsible for erythrocyte binding, two residues in each DBL domain were chosen and mutated to Alanine. In F1, these residues were <scene name='PfEBA-140/Asp247/1'>Asp247</scene> and <scene name='PfEBA-140/Arg254/1'>Arg254</scene>. In F2, these residues were <scene name='PfEBA-140/Arg485/1'>Arg485</scene> and <scene name='PfEBA-140/Asp554/2'>Asp554</scene>. When these four residues were individually mutated to Alanine, erythrocyte binding was heavily interrupted. This can be observed in the figure to the right.<ref name="Lin">PMID: 22989878</ref>[[Image:DecreasedBinding.png | thumb | 100px | alt=text | Mutation of Asp247, Arg254, Arg485, and Asp554]] The decreased erythrocyte binding confirms that these <scene name='PfEBA-140/Four/1'>four residues</scene> found in the basic patch of RII PfEBA-140 are crucial to erythrocyte binding. As a control, residues on the opposite side of RII PfEBA-140 were mutated to Alanine. These residues were Asn394 and Glu605. Mutation of these two residues had no effect on erythrocyte binding.<ref name="Lin">PMID: 22989878</ref>[[Image:FullBinding.png | thumb | 100px | alt=text | Mutation of Asn394 and Glu605]] These results fortify the finding that erythrocyte binding takes place in the basic patch discussed earlier.

 
 
 
 
 

==Structural Differences in PfEBA-140 from other EBL Ligands==
<Structure load='4GF2' size='400' frame='true' align='left' caption='Crystal Structure of PfEBA-140 (PDB entry [[4GF2]])' scene=''/>
There are three unique structural elements that set RII PfEBA-140 apart from other EBL ligands. These structures are the likely reason behind RII PfEBA-140 binding GPC so specifically.
===Disulfide Bonds===
[[Image:ModifiedPattern.png | thumb | 300px | alt=text | Disulfide Bonding Patterns]]
There are twenty-six <scene name='PfEBA-140/Cysteines/1'>cysteines</scene> in RII PfEBA-140, all of which are involved in <scene name='PfEBA-140/Disulfidebonds/1'>disulfide bonding</scene> (disulfide bonds shown in black).<ref name="Lin">PMID: 22989878</ref> In RII PfEBA-140, two modified disulfide patterns were found to be different than in RII PfEBA-175, RII PvDBP, and RII PkDBP. In the latter three RII domains, there is a disulfide linkage between Cys7 and Cys9 and between Cys8 and Cys12. In RII PfEBA-140, however, Cys7 is disulfide bonded to Cys8, and Cys9 is disulfide bonded to Cys12.<ref name="Lin">PMID: 22989878</ref>

===Alpha Helix Structure===
Another difference between RII PfEBA-140 and other EBL ligands can be found in the F2 domain. In EBL ligands of other ''Plasmodium'' species, there are β-fingers with either long or short loops which may or may not be important for receptor binding.<ref name="Lin">PMID: 22989878</ref> While this β-finger is found in F1 of PfEBA-140, it is not found in F2, as it is replaced with an <scene name='PfEBA-140/Alphabeta/1'>α-helix</scene>.<ref name="Lin">PMID: 22989878</ref>
===Lack of Kink in F1===
A third difference between RII PfEBA-140 and other EBL ligands is the lack of a glycine residue in F1. This glycine is present in F1 and F2 of PfEBA-175, PvDBP, PkDBP-α, and F2 PfEBA-140. In F2 PfEBA-140, this glycine is residue 627.<ref name="Lin">PMID: 22989878</ref> In these EBL ligands there is a kink due to glycine's flexibility. If this glycine residue were present in F1 of PfEBA-140, it would result in a kink in subdomain 3.<ref name="Lin">PMID: 22989878</ref> This kink would create a hinge angle between the two DBL domains that would allow for dimerization of PfEBA-140 upon erythrocyte binding. For this reason, PfEBA-140 is believed to engage erythrocytes as a monomer, unlike other EBL ligands, which bind erythrocytes as a dimer.
 
 
These three structural differences are believed to be responsible for the specificity of RII PfEBA-140.

==References==
<references />

File:SubdomainsF1F2.png

2012-11-24T22:21:15Z

Emily Lum: /* Licensing */

== Summary ==
Subdomains S1, S2, and S3 found in F1 and F2.<ref name="Lin">PMID: 22989878</ref>
== Licensing ==
{{subst:Non-commercial from license selector}}

This research was originally published in The Journal of Biological Chemistry. Daniel H. Lin, Brian M. Malpede, Joseph D. Batchelor and Niraj H. Tolia. Crystal and Solution Structures of Plasmodium falciparum Erythrocyte-binding Antigen 140 Reveal Determinants of Receptor Specificity during Erythrocyte Invasion. J Biol Chem. 2012; Vol:287. 36830-36836. © the American Society for Biochemistry and Molecular Biology."

For other parties using material for noncommercial use:

Other parties are welcome to copy, distribute, transmit and adapt the work — at no cost and without permission — for noncommercial use as long as they attribute the work to the original source using the citation above.

Examples of noncommercial use include:

Reproducing a figure for educational purposes, such as schoolwork or lecture presentations, with attribution.
Appending a reprinted article to a Ph.D. dissertation, with attribution.

==References==
<references />

File:ModifiedPattern.png

2012-11-24T22:20:53Z

Emily Lum: /* Licensing */

== Licensing ==
{{subst:Non-commercial from license selector}}

This research was originally published in The Journal of Biological Chemistry. Daniel H. Lin, Brian M. Malpede, Joseph D. Batchelor and Niraj H. Tolia. Crystal and Solution Structures of Plasmodium falciparum Erythrocyte-binding Antigen 140 Reveal Determinants of Receptor Specificity during Erythrocyte Invasion. J Biol Chem. 2012; Vol:287. 36830-36836. © the American Society for Biochemistry and Molecular Biology."

For other parties using material for noncommercial use:

Other parties are welcome to copy, distribute, transmit and adapt the work — at no cost and without permission — for noncommercial use as long as they attribute the work to the original source using the citation above.

Examples of noncommercial use include:

Reproducing a figure for educational purposes, such as schoolwork or lecture presentations, with attribution.
Appending a reprinted article to a Ph.D. dissertation, with attribution.

==References==
<references />

File:IndividualSubdomains.png

2012-11-24T22:20:35Z

Emily Lum: /* Licensing */

== Summary ==
Subdomains of RII PfEBA-140.<ref name="Lin">PMID: 22989878</ref>
== Licensing ==
{{subst:Non-commercial from license selector}}

This research was originally published in The Journal of Biological Chemistry. Daniel H. Lin, Brian M. Malpede, Joseph D. Batchelor and Niraj H. Tolia. Crystal and Solution Structures of Plasmodium falciparum Erythrocyte-binding Antigen 140 Reveal Determinants of Receptor Specificity during Erythrocyte Invasion. J Biol Chem. 2012; Vol:287. 36830-36836. © the American Society for Biochemistry and Molecular Biology."

For other parties using material for noncommercial use:

Other parties are welcome to copy, distribute, transmit and adapt the work — at no cost and without permission — for noncommercial use as long as they attribute the work to the original source using the citation above.

Examples of noncommercial use include:

Reproducing a figure for educational purposes, such as schoolwork or lecture presentations, with attribution.
Appending a reprinted article to a Ph.D. dissertation, with attribution.

==References==
<references />

File:FullBinding.png

2012-11-24T22:20:16Z

Emily Lum: /* Licensing */

File:DecreasedBinding.png

2012-11-24T22:20:00Z

Emily Lum: /* Licensing */

File:Binding.png

2012-11-24T22:19:42Z

Emily Lum: /* Licensing */

== Summary ==
Full length RII of PfEBA-140 binds extensively to erythrocytes (top image), while individual DBL domains show little binding. This indicates that both domains are necessary for erythrocyte binding.<ref name="Lin">PMID: 22989878</ref>
== Licensing ==
{{subst:Non-commercial from license selector}}

This research was originally published in The Journal of Biological Chemistry. Daniel H. Lin, Brian M. Malpede, Joseph D. Batchelor and Niraj H. Tolia. Crystal and Solution Structures of Plasmodium falciparum Erythrocyte-binding Antigen 140 Reveal Determinants of Receptor Specificity during Erythrocyte Invasion. J Biol Chem. 2012; Vol:287. 36830-36836. © the American Society for Biochemistry and Molecular Biology."

For other parties using material for noncommercial use:

Other parties are welcome to copy, distribute, transmit and adapt the work — at no cost and without permission — for noncommercial use as long as they attribute the work to the original source using the citation above.

Examples of noncommercial use include:

Reproducing a figure for educational purposes, such as schoolwork or lecture presentations, with attribution.
Appending a reprinted article to a Ph.D. dissertation, with attribution.

==References==
<references />

File:Basic Patch.png

2012-11-24T22:18:53Z

Emily Lum: /* Licensing */

== Summary ==
Basic patch of RII PfEBA-140.<ref name="Lin">PMID: 22989878</ref>
== Licensing ==
{{subst:Non-commercial from license selector}}

This research was originally published in The Journal of Biological Chemistry. Daniel H. Lin, Brian M. Malpede, Joseph D. Batchelor and Niraj H. Tolia. Crystal and Solution Structures of Plasmodium falciparum Erythrocyte-binding Antigen 140 Reveal Determinants of Receptor Specificity during Erythrocyte Invasion. J Biol Chem. 2012; Vol:287. 36830-36836. © the American Society for Biochemistry and Molecular Biology."

For other parties using material for noncommercial use:

Other parties are welcome to copy, distribute, transmit and adapt the work — at no cost and without permission — for noncommercial use as long as they attribute the work to the original source using the citation above.

Examples of noncommercial use include:

Reproducing a figure for educational purposes, such as schoolwork or lecture presentations, with attribution.
Appending a reprinted article to a Ph.D. dissertation, with attribution.

==References==
<references />

Malaria Parasite Plasmodium falciparum Erythrocyte Binding Antigen 140

2012-11-15T23:52:11Z

Emily Lum: /* Lack of Kink in F1 */

== Introduction ==
In 2010, malaria caused over 650,000 deaths.<ref name="World Health Organization">http://www.who.int/mediacentre/factsheets/fs094/en/index.html</ref> While this disease is both preventable and curable, many of those that live in areas where the disease is endemic do not have access to such resources. Although there is a vaccine currently undergoing clinical trials, results are not expected until 2014.<ref name="World Health Organization">http://www.who.int/mediacentre/factsheets/fs094/en/index.html</ref> The most recent vaccine research has focused on understanding a group of proteins in the erythrocyte-binding ligand (EBL) family. These proteins, which are found in the ''Plasmodium'' species, facilitate entry into erythrocytes during malarial infection by creating tight junctions between the host erythrocytes and parasite membranes. <ref name="Lin">PMID: 22989878</ref> There are four family members: erythrocyte-binding antigen 175 (PfEBA-175), erythrocyte-binding ligand 1 (PfEBL-1), erythrocyte-binding antigen 140 (PfEBA-140), and erythrocyte-binding antigen 181 (PfEBA-181).<ref name="Lin">PMID: 22989878</ref> PfEBA-140 binds glycophorin C on host erythrocytes,<ref name="Lin">PMID: 22989878</ref> which helps maintain erythrocyte shape and regulates membrane material properties.<ref name="Glycophorin C">http://en.wikipedia.org/wiki/Glycophorin_C</ref> Understanding the mechanism by which PfEBA-140 recognizes and engages glycophorin C on erythrocytes may lead to the future development of a new malaria vaccine, as it has been found to be immunogenic.<ref name="Ford">PMID: 17984363</ref>

==General Structure==
<Structure load='4GF2' size='400' frame='true' align='left' caption='Crystal Structure of PfEBA-140 (PDB entry [[4GF2]])' scene=''/>
[[Image:SubdomainsF1F2.png | thumb | 300px | alt=text | Subdomains of F1 and F2]]
[[Image:IndividualSubdomains.png | thumb | 300px | alt=text | Subdomains of F1 and F2]]
The EBL family members, including PfEBA-140, are made up of two regions, region II (RII) (shown to the right) and region VI.<ref name="Lin">PMID: 22989878</ref> Region II is responsible for receptor binding in all EBL family members.<ref name="Lin">PMID: 22989878</ref> RII is composed of <scene name='PfEBA-140/Rii/1'>two Duffy binding-like (DBL) domains</scene>, F1 (purple) and F2 (teal).<ref name="Lin">PMID: 22989878</ref> These two DBL domains are connected by a <scene name='PfEBA-140/Helical_linker/2'>short helical linker</scene>.<ref name="Lin">PMID: 22989878</ref> The DBL protein fold is unique to the ''Plasmodium'' species. Not only does it have the ability to recognize and bind many erythrocyte cell receptors, but it also mediates microvasculature adherence of infected erythrocytes by erythrocyte membrane protein 1 (PfEMP1).<ref name="Lin">PMID: 22989878</ref> Each DBL domain is composed of three subdomains, illustrated in the pictures to the right.

In the top image, the subdomains S1, S2, and S3 of each of the F1 and F2 domains, as well as the helical linker, are illustrated.<ref name="Lin">PMID: 22989878</ref>
In the bottom image, structures of the individual subdomains are illustrated.<ref name="Lin">PMID: 22989878</ref>

The colors are the same in both images: F1 subdomain 1 is shown in bronze, subdomain 2 in orange, subdomain 3 in dark orange; F2 subdomain 1 is shown in dark blue, subdomain 2 in blue, and subdomain 3 in light blue.

==Binding of RII PfEBA-140==
<Structure load='4GF2' size='400' frame='true' align='left' caption='Crystal Structure of PfEBA-140 (PDB entry [[4GF2]])' scene=''/>
===Requirement of Both DBL Domains===
[[Image:Binding.png | thumb | 100px | alt=text | Erythrocyte Binding of Full Length and Individual RII Domains]]Since the DBL domains of RII are highly conserved, the ability of the individual DBL domains to bind erythrocytes was tested. To do this, constructs containing the full-length RII PfEBA-140 and each individual DBL domain were tested using a rosetting assay. Both brightfield microscopy and green fluorescence protein (GFP) were used to visualize erythrocyte binding. In the upper panel, a construct expressing only GFP was used as a control. The lower panel of Figure A illustrates the extensive erythrocyte binding of the full length RII construct. In Figure B, the lack of black dots in the brightfield microscopy and the smaller quantity of green fluorescence illustrates that F1 and F2 are unable to independently bind erythrocytes. This result suggests that both domains equally participate in engaging erythrocytes.<ref name="Lin">PMID: 22989878</ref>
===Basic Patch===
[[Image:Basic Patch.png | thumb | 100px | alt=text | Erythrocyte Binding of Full Length and Individual RII Domains]]
Since the two DBL domains bind acidic GPC, there must be a number of basic charges that interact with the acidic GPC residues. This idea is supported by visualizing the <scene name='PfEBA-140/Basic_patch/1'>positive and negative residues</scene> found in RII PfEBA-140. The positive side chain residues are bue while the negative side chain residues are red. On the side shown, there are a number of residues with positive side chains, creating a basic patch. This basic patch, which forms an arch spanning the two DBL domains, is thought to interact with the acidic GPC, because if the molecule is flipped 180°, there is no concentration of positive charges. The basic patch may be better visualized in the picture to the right.<ref name="Lin">PMID: 22989878</ref>
To test whether this basic patch was indeed responsible for erythrocyte binding, two residues in each DBL domain were chosen and mutated to Alanine. In F1, these residues were <scene name='PfEBA-140/Asp247/1'>Asp247</scene> and <scene name='PfEBA-140/Arg254/1'>Arg254</scene>. In F2, these residues were <scene name='PfEBA-140/Arg485/1'>Arg485</scene> and <scene name='PfEBA-140/Asp554/2'>Asp554</scene>. When these four residues were individually mutated to Alanine, erythrocyte binding was heavily interrupted. This can be observed in the figure to the right.<ref name="Lin">PMID: 22989878</ref>[[Image:DecreasedBinding.png | thumb | 100px | alt=text | Mutation of Asp247, Arg254, Arg485, and Asp554]] The decreased erythrocyte binding confirms that these <scene name='PfEBA-140/Four/1'>four residues</scene> found in the basic patch of RII PfEBA-140 are crucial to erythrocyte binding. As a control, residues on the opposite side of RII PfEBA-140 were mutated to Alanine. These residues were Asn394 and Glu605. Mutation of these two residues had no effect on erythrocyte binding.<ref name="Lin">PMID: 22989878</ref>[[Image:FullBinding.png | thumb | 100px | alt=text | Mutation of Asn394 and Glu605]] These results fortify the finding that erythrocyte binding takes place in the basic patch discussed earlier.

 
 
 
 
 

==Structural Differences in PfEBA-140 from other EBL Ligands==
<Structure load='4GF2' size='400' frame='true' align='left' caption='Crystal Structure of PfEBA-140 (PDB entry [[4GF2]])' scene=''/>
There are three unique structural elements that set RII PfEBA-140 apart from other EBL ligands. These structures are the likely reason behind RII PfEBA-140 binding GPC so specifically.
===Disulfide Bonds===
[[Image:ModifiedPattern.png | thumb | 300px | alt=text | Disulfide Bonding Patterns]]
There are twenty-six <scene name='PfEBA-140/Cysteines/1'>cysteines</scene> in RII PfEBA-140, all of which are involved in <scene name='PfEBA-140/Disulfidebonds/1'>disulfide bonding</scene> (disulfide bonds shown in black).<ref name="Lin">PMID: 22989878</ref> In RII PfEBA-140, two modified disulfide patterns were found to be different than in RII PfEBA-175, RII PvDBP, and RII PkDBP. In the latter three RII domains, there is a disulfide linkage between Cys7 and Cys9 and between Cys8 and Cys12. In RII PfEBA-140, however, Cys7 is disulfide bonded to Cys8, and Cys9 is disulfide bonded to Cys12.<ref name="Lin">PMID: 22989878</ref>

===Alpha Helix Structure===
Another difference between RII PfEBA-140 and other EBL ligands can be found in the F2 domain. In EBL ligands of other ''Plasmodium'' species, there are β-fingers with either long or short loops which may or may not be important for receptor binding.<ref name="Lin">PMID: 22989878</ref> While this β-finger is found in F1 of PfEBA-140, it is not found in F2, as it is replaced with an <scene name='PfEBA-140/Alphabeta/1'>α-helix</scene>.<ref name="Lin">PMID: 22989878</ref>
===Lack of Kink in F1===
A third difference between RII PfEBA-140 and other EBL ligands is the lack of a glycine residue in F1. This glycine is present in F1 and F2 of PfEBA-175, PvDBP, PkDBP-α, and F2 PfEBA-140. In F2 PfEBA-140, this glycine is residue 627.<ref name="Lin">PMID: 22989878</ref> In these EBL ligands there is a kink due to glycine's flexibility. If this glycine residue were present in F1 of PfEBA-140, it would result in a kink in subdomain 3.<ref name="Lin">PMID: 22989878</ref> This kink would create a hinge angle between the two DBL domains that would allow for dimerization of PfEBA-140 upon erythrocyte binding. For this reason, PfEBA-140 is believed to engage erythrocytes as a monomer, unlike other EBL ligands, which bind erythrocytes as a dimer.
 
 
These three structural differences are believed to be responsible for the specificity of RII PfEBA-140.

==References==
<references />

Malaria Parasite Plasmodium falciparum Erythrocyte Binding Antigen 140

2012-11-15T23:51:18Z

Emily Lum: /* Lack of Kink in F1 */

== Introduction ==
In 2010, malaria caused over 650,000 deaths.<ref name="World Health Organization">http://www.who.int/mediacentre/factsheets/fs094/en/index.html</ref> While this disease is both preventable and curable, many of those that live in areas where the disease is endemic do not have access to such resources. Although there is a vaccine currently undergoing clinical trials, results are not expected until 2014.<ref name="World Health Organization">http://www.who.int/mediacentre/factsheets/fs094/en/index.html</ref> The most recent vaccine research has focused on understanding a group of proteins in the erythrocyte-binding ligand (EBL) family. These proteins, which are found in the ''Plasmodium'' species, facilitate entry into erythrocytes during malarial infection by creating tight junctions between the host erythrocytes and parasite membranes. <ref name="Lin">PMID: 22989878</ref> There are four family members: erythrocyte-binding antigen 175 (PfEBA-175), erythrocyte-binding ligand 1 (PfEBL-1), erythrocyte-binding antigen 140 (PfEBA-140), and erythrocyte-binding antigen 181 (PfEBA-181).<ref name="Lin">PMID: 22989878</ref> PfEBA-140 binds glycophorin C on host erythrocytes,<ref name="Lin">PMID: 22989878</ref> which helps maintain erythrocyte shape and regulates membrane material properties.<ref name="Glycophorin C">http://en.wikipedia.org/wiki/Glycophorin_C</ref> Understanding the mechanism by which PfEBA-140 recognizes and engages glycophorin C on erythrocytes may lead to the future development of a new malaria vaccine, as it has been found to be immunogenic.<ref name="Ford">PMID: 17984363</ref>

==General Structure==
<Structure load='4GF2' size='400' frame='true' align='left' caption='Crystal Structure of PfEBA-140 (PDB entry [[4GF2]])' scene=''/>
[[Image:SubdomainsF1F2.png | thumb | 300px | alt=text | Subdomains of F1 and F2]]
[[Image:IndividualSubdomains.png | thumb | 300px | alt=text | Subdomains of F1 and F2]]
The EBL family members, including PfEBA-140, are made up of two regions, region II (RII) (shown to the right) and region VI.<ref name="Lin">PMID: 22989878</ref> Region II is responsible for receptor binding in all EBL family members.<ref name="Lin">PMID: 22989878</ref> RII is composed of <scene name='PfEBA-140/Rii/1'>two Duffy binding-like (DBL) domains</scene>, F1 (purple) and F2 (teal).<ref name="Lin">PMID: 22989878</ref> These two DBL domains are connected by a <scene name='PfEBA-140/Helical_linker/2'>short helical linker</scene>.<ref name="Lin">PMID: 22989878</ref> The DBL protein fold is unique to the ''Plasmodium'' species. Not only does it have the ability to recognize and bind many erythrocyte cell receptors, but it also mediates microvasculature adherence of infected erythrocytes by erythrocyte membrane protein 1 (PfEMP1).<ref name="Lin">PMID: 22989878</ref> Each DBL domain is composed of three subdomains, illustrated in the pictures to the right.

In the top image, the subdomains S1, S2, and S3 of each of the F1 and F2 domains, as well as the helical linker, are illustrated.<ref name="Lin">PMID: 22989878</ref>
In the bottom image, structures of the individual subdomains are illustrated.<ref name="Lin">PMID: 22989878</ref>

The colors are the same in both images: F1 subdomain 1 is shown in bronze, subdomain 2 in orange, subdomain 3 in dark orange; F2 subdomain 1 is shown in dark blue, subdomain 2 in blue, and subdomain 3 in light blue.

==Binding of RII PfEBA-140==
<Structure load='4GF2' size='400' frame='true' align='left' caption='Crystal Structure of PfEBA-140 (PDB entry [[4GF2]])' scene=''/>
===Requirement of Both DBL Domains===
[[Image:Binding.png | thumb | 100px | alt=text | Erythrocyte Binding of Full Length and Individual RII Domains]]Since the DBL domains of RII are highly conserved, the ability of the individual DBL domains to bind erythrocytes was tested. To do this, constructs containing the full-length RII PfEBA-140 and each individual DBL domain were tested using a rosetting assay. Both brightfield microscopy and green fluorescence protein (GFP) were used to visualize erythrocyte binding. In the upper panel, a construct expressing only GFP was used as a control. The lower panel of Figure A illustrates the extensive erythrocyte binding of the full length RII construct. In Figure B, the lack of black dots in the brightfield microscopy and the smaller quantity of green fluorescence illustrates that F1 and F2 are unable to independently bind erythrocytes. This result suggests that both domains equally participate in engaging erythrocytes.<ref name="Lin">PMID: 22989878</ref>
===Basic Patch===
[[Image:Basic Patch.png | thumb | 100px | alt=text | Erythrocyte Binding of Full Length and Individual RII Domains]]
Since the two DBL domains bind acidic GPC, there must be a number of basic charges that interact with the acidic GPC residues. This idea is supported by visualizing the <scene name='PfEBA-140/Basic_patch/1'>positive and negative residues</scene> found in RII PfEBA-140. The positive side chain residues are bue while the negative side chain residues are red. On the side shown, there are a number of residues with positive side chains, creating a basic patch. This basic patch, which forms an arch spanning the two DBL domains, is thought to interact with the acidic GPC, because if the molecule is flipped 180°, there is no concentration of positive charges. The basic patch may be better visualized in the picture to the right.<ref name="Lin">PMID: 22989878</ref>
To test whether this basic patch was indeed responsible for erythrocyte binding, two residues in each DBL domain were chosen and mutated to Alanine. In F1, these residues were <scene name='PfEBA-140/Asp247/1'>Asp247</scene> and <scene name='PfEBA-140/Arg254/1'>Arg254</scene>. In F2, these residues were <scene name='PfEBA-140/Arg485/1'>Arg485</scene> and <scene name='PfEBA-140/Asp554/2'>Asp554</scene>. When these four residues were individually mutated to Alanine, erythrocyte binding was heavily interrupted. This can be observed in the figure to the right.<ref name="Lin">PMID: 22989878</ref>[[Image:DecreasedBinding.png | thumb | 100px | alt=text | Mutation of Asp247, Arg254, Arg485, and Asp554]] The decreased erythrocyte binding confirms that these <scene name='PfEBA-140/Four/1'>four residues</scene> found in the basic patch of RII PfEBA-140 are crucial to erythrocyte binding. As a control, residues on the opposite side of RII PfEBA-140 were mutated to Alanine. These residues were Asn394 and Glu605. Mutation of these two residues had no effect on erythrocyte binding.<ref name="Lin">PMID: 22989878</ref>[[Image:FullBinding.png | thumb | 100px | alt=text | Mutation of Asn394 and Glu605]] These results fortify the finding that erythrocyte binding takes place in the basic patch discussed earlier.

 
 
 
 
 

==Structural Differences in PfEBA-140 from other EBL Ligands==
<Structure load='4GF2' size='400' frame='true' align='left' caption='Crystal Structure of PfEBA-140 (PDB entry [[4GF2]])' scene=''/>
There are three unique structural elements that set RII PfEBA-140 apart from other EBL ligands. These structures are the likely reason behind RII PfEBA-140 binding GPC so specifically.
===Disulfide Bonds===
[[Image:ModifiedPattern.png | thumb | 300px | alt=text | Disulfide Bonding Patterns]]
There are twenty-six <scene name='PfEBA-140/Cysteines/1'>cysteines</scene> in RII PfEBA-140, all of which are involved in <scene name='PfEBA-140/Disulfidebonds/1'>disulfide bonding</scene> (disulfide bonds shown in black).<ref name="Lin">PMID: 22989878</ref> In RII PfEBA-140, two modified disulfide patterns were found to be different than in RII PfEBA-175, RII PvDBP, and RII PkDBP. In the latter three RII domains, there is a disulfide linkage between Cys7 and Cys9 and between Cys8 and Cys12. In RII PfEBA-140, however, Cys7 is disulfide bonded to Cys8, and Cys9 is disulfide bonded to Cys12.<ref name="Lin">PMID: 22989878</ref>

===Alpha Helix Structure===
Another difference between RII PfEBA-140 and other EBL ligands can be found in the F2 domain. In EBL ligands of other ''Plasmodium'' species, there are β-fingers with either long or short loops which may or may not be important for receptor binding.<ref name="Lin">PMID: 22989878</ref> While this β-finger is found in F1 of PfEBA-140, it is not found in F2, as it is replaced with an <scene name='PfEBA-140/Alphabeta/1'>α-helix</scene>.<ref name="Lin">PMID: 22989878</ref>
===Lack of Kink in F1===
A third difference between RII PfEBA-140 and other EBL ligands is the lack of a glycine residue in F1. This glycine is present in F1 and F2 of PfEBA-175, PvDBP, PkDBP-α, and F2 PfEBA-140. In F2 PfEBA-140, this glycine is residue 627.<ref name="Lin">PMID: 22989878</ref> In these EBL ligands create a kink due to glycine's flexibility. If this glycine residue were present in F1 of PfEBA-140, it would result in a kink in subdomain 3.<ref name="Lin">PMID: 22989878</ref> This kink would create a hinge angle between the two DBL domains that would allow for dimerization of PfEBA-140 upon erythrocyte binding. For this reason, PfEBA-140 is believed to engage erythrocytes as a monomer, unlike other EBL ligands, which bind erythrocytes as a dimer.
 
 
These three structural differences are believed to be responsible for the specificity of RII PfEBA-140.

==References==
<references />

Malaria Parasite Plasmodium falciparum Erythrocyte Binding Antigen 140

2012-11-15T23:48:32Z

Emily Lum: /* Disulfide Bonds */

== Introduction ==
In 2010, malaria caused over 650,000 deaths.<ref name="World Health Organization">http://www.who.int/mediacentre/factsheets/fs094/en/index.html</ref> While this disease is both preventable and curable, many of those that live in areas where the disease is endemic do not have access to such resources. Although there is a vaccine currently undergoing clinical trials, results are not expected until 2014.<ref name="World Health Organization">http://www.who.int/mediacentre/factsheets/fs094/en/index.html</ref> The most recent vaccine research has focused on understanding a group of proteins in the erythrocyte-binding ligand (EBL) family. These proteins, which are found in the ''Plasmodium'' species, facilitate entry into erythrocytes during malarial infection by creating tight junctions between the host erythrocytes and parasite membranes. <ref name="Lin">PMID: 22989878</ref> There are four family members: erythrocyte-binding antigen 175 (PfEBA-175), erythrocyte-binding ligand 1 (PfEBL-1), erythrocyte-binding antigen 140 (PfEBA-140), and erythrocyte-binding antigen 181 (PfEBA-181).<ref name="Lin">PMID: 22989878</ref> PfEBA-140 binds glycophorin C on host erythrocytes,<ref name="Lin">PMID: 22989878</ref> which helps maintain erythrocyte shape and regulates membrane material properties.<ref name="Glycophorin C">http://en.wikipedia.org/wiki/Glycophorin_C</ref> Understanding the mechanism by which PfEBA-140 recognizes and engages glycophorin C on erythrocytes may lead to the future development of a new malaria vaccine, as it has been found to be immunogenic.<ref name="Ford">PMID: 17984363</ref>

==General Structure==
<Structure load='4GF2' size='400' frame='true' align='left' caption='Crystal Structure of PfEBA-140 (PDB entry [[4GF2]])' scene=''/>
[[Image:SubdomainsF1F2.png | thumb | 300px | alt=text | Subdomains of F1 and F2]]
[[Image:IndividualSubdomains.png | thumb | 300px | alt=text | Subdomains of F1 and F2]]
The EBL family members, including PfEBA-140, are made up of two regions, region II (RII) (shown to the right) and region VI.<ref name="Lin">PMID: 22989878</ref> Region II is responsible for receptor binding in all EBL family members.<ref name="Lin">PMID: 22989878</ref> RII is composed of <scene name='PfEBA-140/Rii/1'>two Duffy binding-like (DBL) domains</scene>, F1 (purple) and F2 (teal).<ref name="Lin">PMID: 22989878</ref> These two DBL domains are connected by a <scene name='PfEBA-140/Helical_linker/2'>short helical linker</scene>.<ref name="Lin">PMID: 22989878</ref> The DBL protein fold is unique to the ''Plasmodium'' species. Not only does it have the ability to recognize and bind many erythrocyte cell receptors, but it also mediates microvasculature adherence of infected erythrocytes by erythrocyte membrane protein 1 (PfEMP1).<ref name="Lin">PMID: 22989878</ref> Each DBL domain is composed of three subdomains, illustrated in the pictures to the right.

In the top image, the subdomains S1, S2, and S3 of each of the F1 and F2 domains, as well as the helical linker, are illustrated.<ref name="Lin">PMID: 22989878</ref>
In the bottom image, structures of the individual subdomains are illustrated.<ref name="Lin">PMID: 22989878</ref>

The colors are the same in both images: F1 subdomain 1 is shown in bronze, subdomain 2 in orange, subdomain 3 in dark orange; F2 subdomain 1 is shown in dark blue, subdomain 2 in blue, and subdomain 3 in light blue.

==Binding of RII PfEBA-140==
<Structure load='4GF2' size='400' frame='true' align='left' caption='Crystal Structure of PfEBA-140 (PDB entry [[4GF2]])' scene=''/>
===Requirement of Both DBL Domains===
[[Image:Binding.png | thumb | 100px | alt=text | Erythrocyte Binding of Full Length and Individual RII Domains]]Since the DBL domains of RII are highly conserved, the ability of the individual DBL domains to bind erythrocytes was tested. To do this, constructs containing the full-length RII PfEBA-140 and each individual DBL domain were tested using a rosetting assay. Both brightfield microscopy and green fluorescence protein (GFP) were used to visualize erythrocyte binding. In the upper panel, a construct expressing only GFP was used as a control. The lower panel of Figure A illustrates the extensive erythrocyte binding of the full length RII construct. In Figure B, the lack of black dots in the brightfield microscopy and the smaller quantity of green fluorescence illustrates that F1 and F2 are unable to independently bind erythrocytes. This result suggests that both domains equally participate in engaging erythrocytes.<ref name="Lin">PMID: 22989878</ref>
===Basic Patch===
[[Image:Basic Patch.png | thumb | 100px | alt=text | Erythrocyte Binding of Full Length and Individual RII Domains]]
Since the two DBL domains bind acidic GPC, there must be a number of basic charges that interact with the acidic GPC residues. This idea is supported by visualizing the <scene name='PfEBA-140/Basic_patch/1'>positive and negative residues</scene> found in RII PfEBA-140. The positive side chain residues are bue while the negative side chain residues are red. On the side shown, there are a number of residues with positive side chains, creating a basic patch. This basic patch, which forms an arch spanning the two DBL domains, is thought to interact with the acidic GPC, because if the molecule is flipped 180°, there is no concentration of positive charges. The basic patch may be better visualized in the picture to the right.<ref name="Lin">PMID: 22989878</ref>
To test whether this basic patch was indeed responsible for erythrocyte binding, two residues in each DBL domain were chosen and mutated to Alanine. In F1, these residues were <scene name='PfEBA-140/Asp247/1'>Asp247</scene> and <scene name='PfEBA-140/Arg254/1'>Arg254</scene>. In F2, these residues were <scene name='PfEBA-140/Arg485/1'>Arg485</scene> and <scene name='PfEBA-140/Asp554/2'>Asp554</scene>. When these four residues were individually mutated to Alanine, erythrocyte binding was heavily interrupted. This can be observed in the figure to the right.<ref name="Lin">PMID: 22989878</ref>[[Image:DecreasedBinding.png | thumb | 100px | alt=text | Mutation of Asp247, Arg254, Arg485, and Asp554]] The decreased erythrocyte binding confirms that these <scene name='PfEBA-140/Four/1'>four residues</scene> found in the basic patch of RII PfEBA-140 are crucial to erythrocyte binding. As a control, residues on the opposite side of RII PfEBA-140 were mutated to Alanine. These residues were Asn394 and Glu605. Mutation of these two residues had no effect on erythrocyte binding.<ref name="Lin">PMID: 22989878</ref>[[Image:FullBinding.png | thumb | 100px | alt=text | Mutation of Asn394 and Glu605]] These results fortify the finding that erythrocyte binding takes place in the basic patch discussed earlier.

 
 
 
 
 

==Structural Differences in PfEBA-140 from other EBL Ligands==
<Structure load='4GF2' size='400' frame='true' align='left' caption='Crystal Structure of PfEBA-140 (PDB entry [[4GF2]])' scene=''/>
There are three unique structural elements that set RII PfEBA-140 apart from other EBL ligands. These structures are the likely reason behind RII PfEBA-140 binding GPC so specifically.
===Disulfide Bonds===
[[Image:ModifiedPattern.png | thumb | 300px | alt=text | Disulfide Bonding Patterns]]
There are twenty-six <scene name='PfEBA-140/Cysteines/1'>cysteines</scene> in RII PfEBA-140, all of which are involved in <scene name='PfEBA-140/Disulfidebonds/1'>disulfide bonding</scene> (disulfide bonds shown in black).<ref name="Lin">PMID: 22989878</ref> In RII PfEBA-140, two modified disulfide patterns were found to be different than in RII PfEBA-175, RII PvDBP, and RII PkDBP. In the latter three RII domains, there is a disulfide linkage between Cys7 and Cys9 and between Cys8 and Cys12. In RII PfEBA-140, however, Cys7 is disulfide bonded to Cys8, and Cys9 is disulfide bonded to Cys12.<ref name="Lin">PMID: 22989878</ref>

===Alpha Helix Structure===
Another difference between RII PfEBA-140 and other EBL ligands can be found in the F2 domain. In EBL ligands of other ''Plasmodium'' species, there are β-fingers with either long or short loops which may or may not be important for receptor binding.<ref name="Lin">PMID: 22989878</ref> While this β-finger is found in F1 of PfEBA-140, it is not found in F2, as it is replaced with an <scene name='PfEBA-140/Alphabeta/1'>α-helix</scene>.<ref name="Lin">PMID: 22989878</ref>
===Lack of Kink in F1===
A third difference between RII PfEBA-140 and other EBL ligands is the lack of a glycine residue in F1. This glycine is present in F1 PfEBA-175, F2 PfEBA-175, PvDBP, PkDBP-α, and F2 PfEBA-140. In F2 PfEBA-140, this glycine is residue 627.<ref name="Lin">PMID: 22989878</ref> In these EBL ligands create a kink due to glycine's flexibility. If this glycine residue were present in F1 of PfEBA-140, it would result in a kink in subdomain 3.<ref name="Lin">PMID: 22989878</ref> This kink would create a hinge angle between the two DBL domains that would allow for dimerization of PfEBA-140 upon erythrocyte binding. For this reason, PfEBA-140 is believed to engage erythrocytes as a monomer, unlike other EBL ligands, which bind erythrocytes as a dimer.
 
 
These three structural differences are believed to be responsible for the specificity of RII PfEBA-140.

==References==
<references />

Malaria Parasite Plasmodium falciparum Erythrocyte Binding Antigen 140

2012-11-15T23:42:16Z

Emily Lum: /* Basic Patch */

== Introduction ==
In 2010, malaria caused over 650,000 deaths.<ref name="World Health Organization">http://www.who.int/mediacentre/factsheets/fs094/en/index.html</ref> While this disease is both preventable and curable, many of those that live in areas where the disease is endemic do not have access to such resources. Although there is a vaccine currently undergoing clinical trials, results are not expected until 2014.<ref name="World Health Organization">http://www.who.int/mediacentre/factsheets/fs094/en/index.html</ref> The most recent vaccine research has focused on understanding a group of proteins in the erythrocyte-binding ligand (EBL) family. These proteins, which are found in the ''Plasmodium'' species, facilitate entry into erythrocytes during malarial infection by creating tight junctions between the host erythrocytes and parasite membranes. <ref name="Lin">PMID: 22989878</ref> There are four family members: erythrocyte-binding antigen 175 (PfEBA-175), erythrocyte-binding ligand 1 (PfEBL-1), erythrocyte-binding antigen 140 (PfEBA-140), and erythrocyte-binding antigen 181 (PfEBA-181).<ref name="Lin">PMID: 22989878</ref> PfEBA-140 binds glycophorin C on host erythrocytes,<ref name="Lin">PMID: 22989878</ref> which helps maintain erythrocyte shape and regulates membrane material properties.<ref name="Glycophorin C">http://en.wikipedia.org/wiki/Glycophorin_C</ref> Understanding the mechanism by which PfEBA-140 recognizes and engages glycophorin C on erythrocytes may lead to the future development of a new malaria vaccine, as it has been found to be immunogenic.<ref name="Ford">PMID: 17984363</ref>

==General Structure==
<Structure load='4GF2' size='400' frame='true' align='left' caption='Crystal Structure of PfEBA-140 (PDB entry [[4GF2]])' scene=''/>
[[Image:SubdomainsF1F2.png | thumb | 300px | alt=text | Subdomains of F1 and F2]]
[[Image:IndividualSubdomains.png | thumb | 300px | alt=text | Subdomains of F1 and F2]]
The EBL family members, including PfEBA-140, are made up of two regions, region II (RII) (shown to the right) and region VI.<ref name="Lin">PMID: 22989878</ref> Region II is responsible for receptor binding in all EBL family members.<ref name="Lin">PMID: 22989878</ref> RII is composed of <scene name='PfEBA-140/Rii/1'>two Duffy binding-like (DBL) domains</scene>, F1 (purple) and F2 (teal).<ref name="Lin">PMID: 22989878</ref> These two DBL domains are connected by a <scene name='PfEBA-140/Helical_linker/2'>short helical linker</scene>.<ref name="Lin">PMID: 22989878</ref> The DBL protein fold is unique to the ''Plasmodium'' species. Not only does it have the ability to recognize and bind many erythrocyte cell receptors, but it also mediates microvasculature adherence of infected erythrocytes by erythrocyte membrane protein 1 (PfEMP1).<ref name="Lin">PMID: 22989878</ref> Each DBL domain is composed of three subdomains, illustrated in the pictures to the right.

In the top image, the subdomains S1, S2, and S3 of each of the F1 and F2 domains, as well as the helical linker, are illustrated.<ref name="Lin">PMID: 22989878</ref>
In the bottom image, structures of the individual subdomains are illustrated.<ref name="Lin">PMID: 22989878</ref>

The colors are the same in both images: F1 subdomain 1 is shown in bronze, subdomain 2 in orange, subdomain 3 in dark orange; F2 subdomain 1 is shown in dark blue, subdomain 2 in blue, and subdomain 3 in light blue.

==Binding of RII PfEBA-140==
<Structure load='4GF2' size='400' frame='true' align='left' caption='Crystal Structure of PfEBA-140 (PDB entry [[4GF2]])' scene=''/>
===Requirement of Both DBL Domains===
[[Image:Binding.png | thumb | 100px | alt=text | Erythrocyte Binding of Full Length and Individual RII Domains]]Since the DBL domains of RII are highly conserved, the ability of the individual DBL domains to bind erythrocytes was tested. To do this, constructs containing the full-length RII PfEBA-140 and each individual DBL domain were tested using a rosetting assay. Both brightfield microscopy and green fluorescence protein (GFP) were used to visualize erythrocyte binding. In the upper panel, a construct expressing only GFP was used as a control. The lower panel of Figure A illustrates the extensive erythrocyte binding of the full length RII construct. In Figure B, the lack of black dots in the brightfield microscopy and the smaller quantity of green fluorescence illustrates that F1 and F2 are unable to independently bind erythrocytes. This result suggests that both domains equally participate in engaging erythrocytes.<ref name="Lin">PMID: 22989878</ref>
===Basic Patch===
[[Image:Basic Patch.png | thumb | 100px | alt=text | Erythrocyte Binding of Full Length and Individual RII Domains]]
Since the two DBL domains bind acidic GPC, there must be a number of basic charges that interact with the acidic GPC residues. This idea is supported by visualizing the <scene name='PfEBA-140/Basic_patch/1'>positive and negative residues</scene> found in RII PfEBA-140. The positive side chain residues are bue while the negative side chain residues are red. On the side shown, there are a number of residues with positive side chains, creating a basic patch. This basic patch, which forms an arch spanning the two DBL domains, is thought to interact with the acidic GPC, because if the molecule is flipped 180°, there is no concentration of positive charges. The basic patch may be better visualized in the picture to the right.<ref name="Lin">PMID: 22989878</ref>
To test whether this basic patch was indeed responsible for erythrocyte binding, two residues in each DBL domain were chosen and mutated to Alanine. In F1, these residues were <scene name='PfEBA-140/Asp247/1'>Asp247</scene> and <scene name='PfEBA-140/Arg254/1'>Arg254</scene>. In F2, these residues were <scene name='PfEBA-140/Arg485/1'>Arg485</scene> and <scene name='PfEBA-140/Asp554/2'>Asp554</scene>. When these four residues were individually mutated to Alanine, erythrocyte binding was heavily interrupted. This can be observed in the figure to the right.<ref name="Lin">PMID: 22989878</ref>[[Image:DecreasedBinding.png | thumb | 100px | alt=text | Mutation of Asp247, Arg254, Arg485, and Asp554]] The decreased erythrocyte binding confirms that these <scene name='PfEBA-140/Four/1'>four residues</scene> found in the basic patch of RII PfEBA-140 are crucial to erythrocyte binding. As a control, residues on the opposite side of RII PfEBA-140 were mutated to Alanine. These residues were Asn394 and Glu605. Mutation of these two residues had no effect on erythrocyte binding.<ref name="Lin">PMID: 22989878</ref>[[Image:FullBinding.png | thumb | 100px | alt=text | Mutation of Asn394 and Glu605]] These results fortify the finding that erythrocyte binding takes place in the basic patch discussed earlier.

 
 
 
 
 

==Structural Differences in PfEBA-140 from other EBL Ligands==
<Structure load='4GF2' size='400' frame='true' align='left' caption='Crystal Structure of PfEBA-140 (PDB entry [[4GF2]])' scene=''/>
There are three unique structural elements that set RII PfEBA-140 apart from other EBL ligands. These structures are the likely reason behind RII PfEBA-140 binding GPC so specifically.
===Disulfide Bonds===
[[Image:ModifiedPattern.png | thumb | 300px | alt=text | Disulfide Bonding Patterns]]
There are twenty-six <scene name='PfEBA-140/Cysteines/1'>cysteines</scene> in RII PfEBA-140, all of which are involved in <scene name='PfEBA-140/Disulfidebonds/1'>disulfide bonding</scene> (disulfide bonds shown in black).<ref name="Lin">PMID: 22989878</ref> In RII PfEBA-140, two modified disulfide patterns were found to be different than in RII PfEBA-175, RII PvDBP, and RII PkDBP. In the latter three RII domains, there is a disulfide linkage between Cys7 and Cys9 and between Cys8 and Cys12. In RII PfEBA-140, however, Cys7 is disulfide bonded to Cys8 and Cys9 is disulfide bonded to Cys12.<ref name="Lin">PMID: 22989878</ref>
===Alpha Helix Structure===
Another difference between RII PfEBA-140 and other EBL ligands can be found in the F2 domain. In EBL ligands of other ''Plasmodium'' species, there are β-fingers with either long or short loops which may or may not be important for receptor binding.<ref name="Lin">PMID: 22989878</ref> While this β-finger is found in F1 of PfEBA-140, it is not found in F2, as it is replaced with an <scene name='PfEBA-140/Alphabeta/1'>α-helix</scene>.<ref name="Lin">PMID: 22989878</ref>
===Lack of Kink in F1===
A third difference between RII PfEBA-140 and other EBL ligands is the lack of a glycine residue in F1. This glycine is present in F1 PfEBA-175, F2 PfEBA-175, PvDBP, PkDBP-α, and F2 PfEBA-140. In F2 PfEBA-140, this glycine is residue 627.<ref name="Lin">PMID: 22989878</ref> In these EBL ligands create a kink due to glycine's flexibility. If this glycine residue were present in F1 of PfEBA-140, it would result in a kink in subdomain 3.<ref name="Lin">PMID: 22989878</ref> This kink would create a hinge angle between the two DBL domains that would allow for dimerization of PfEBA-140 upon erythrocyte binding. For this reason, PfEBA-140 is believed to engage erythrocytes as a monomer, unlike other EBL ligands, which bind erythrocytes as a dimer.
 
 
These three structural differences are believed to be responsible for the specificity of RII PfEBA-140.

==References==
<references />

Malaria Parasite Plasmodium falciparum Erythrocyte Binding Antigen 140

2012-11-15T23:41:05Z

Emily Lum: /* Basic Patch */

== Introduction ==
In 2010, malaria caused over 650,000 deaths.<ref name="World Health Organization">http://www.who.int/mediacentre/factsheets/fs094/en/index.html</ref> While this disease is both preventable and curable, many of those that live in areas where the disease is endemic do not have access to such resources. Although there is a vaccine currently undergoing clinical trials, results are not expected until 2014.<ref name="World Health Organization">http://www.who.int/mediacentre/factsheets/fs094/en/index.html</ref> The most recent vaccine research has focused on understanding a group of proteins in the erythrocyte-binding ligand (EBL) family. These proteins, which are found in the ''Plasmodium'' species, facilitate entry into erythrocytes during malarial infection by creating tight junctions between the host erythrocytes and parasite membranes. <ref name="Lin">PMID: 22989878</ref> There are four family members: erythrocyte-binding antigen 175 (PfEBA-175), erythrocyte-binding ligand 1 (PfEBL-1), erythrocyte-binding antigen 140 (PfEBA-140), and erythrocyte-binding antigen 181 (PfEBA-181).<ref name="Lin">PMID: 22989878</ref> PfEBA-140 binds glycophorin C on host erythrocytes,<ref name="Lin">PMID: 22989878</ref> which helps maintain erythrocyte shape and regulates membrane material properties.<ref name="Glycophorin C">http://en.wikipedia.org/wiki/Glycophorin_C</ref> Understanding the mechanism by which PfEBA-140 recognizes and engages glycophorin C on erythrocytes may lead to the future development of a new malaria vaccine, as it has been found to be immunogenic.<ref name="Ford">PMID: 17984363</ref>

==General Structure==
<Structure load='4GF2' size='400' frame='true' align='left' caption='Crystal Structure of PfEBA-140 (PDB entry [[4GF2]])' scene=''/>
[[Image:SubdomainsF1F2.png | thumb | 300px | alt=text | Subdomains of F1 and F2]]
[[Image:IndividualSubdomains.png | thumb | 300px | alt=text | Subdomains of F1 and F2]]
The EBL family members, including PfEBA-140, are made up of two regions, region II (RII) (shown to the right) and region VI.<ref name="Lin">PMID: 22989878</ref> Region II is responsible for receptor binding in all EBL family members.<ref name="Lin">PMID: 22989878</ref> RII is composed of <scene name='PfEBA-140/Rii/1'>two Duffy binding-like (DBL) domains</scene>, F1 (purple) and F2 (teal).<ref name="Lin">PMID: 22989878</ref> These two DBL domains are connected by a <scene name='PfEBA-140/Helical_linker/2'>short helical linker</scene>.<ref name="Lin">PMID: 22989878</ref> The DBL protein fold is unique to the ''Plasmodium'' species. Not only does it have the ability to recognize and bind many erythrocyte cell receptors, but it also mediates microvasculature adherence of infected erythrocytes by erythrocyte membrane protein 1 (PfEMP1).<ref name="Lin">PMID: 22989878</ref> Each DBL domain is composed of three subdomains, illustrated in the pictures to the right.

In the top image, the subdomains S1, S2, and S3 of each of the F1 and F2 domains, as well as the helical linker, are illustrated.<ref name="Lin">PMID: 22989878</ref>
In the bottom image, structures of the individual subdomains are illustrated.<ref name="Lin">PMID: 22989878</ref>

The colors are the same in both images: F1 subdomain 1 is shown in bronze, subdomain 2 in orange, subdomain 3 in dark orange; F2 subdomain 1 is shown in dark blue, subdomain 2 in blue, and subdomain 3 in light blue.

==Binding of RII PfEBA-140==
<Structure load='4GF2' size='400' frame='true' align='left' caption='Crystal Structure of PfEBA-140 (PDB entry [[4GF2]])' scene=''/>
===Requirement of Both DBL Domains===
[[Image:Binding.png | thumb | 100px | alt=text | Erythrocyte Binding of Full Length and Individual RII Domains]]Since the DBL domains of RII are highly conserved, the ability of the individual DBL domains to bind erythrocytes was tested. To do this, constructs containing the full-length RII PfEBA-140 and each individual DBL domain were tested using a rosetting assay. Both brightfield microscopy and green fluorescence protein (GFP) were used to visualize erythrocyte binding. In the upper panel, a construct expressing only GFP was used as a control. The lower panel of Figure A illustrates the extensive erythrocyte binding of the full length RII construct. In Figure B, the lack of black dots in the brightfield microscopy and the smaller quantity of green fluorescence illustrates that F1 and F2 are unable to independently bind erythrocytes. This result suggests that both domains equally participate in engaging erythrocytes.<ref name="Lin">PMID: 22989878</ref>
===Basic Patch===
[[Image:Basic Patch.png | thumb | 100px | alt=text | Erythrocyte Binding of Full Length and Individual RII Domains]]
Since the two DBL domains bind acidic GPC, there must be a number of basic charges that interact with the acidic GPC residues. This idea is supported by visualizing the <scene name='PfEBA-140/Basic_patch/1'>positive and negative residues</scene> found in RII PfEBA-140. The positive side chain residues are red while the negative side chain residues are blue. On the side shown, there are a number of residues with positive side chains, creating a basic patch. This basic patch, which forms an arch spanning the two DBL domains, is thought to interact with the acidic GPC, because if the molecule is flipped 180°, there is no concentration of positive charges. The basic patch may be better visualized in the picture to the right.<ref name="Lin">PMID: 22989878</ref>
To test whether this basic patch was indeed responsible for erythrocyte binding, two residues in each DBL domain were chosen and mutated to Alanine. In F1, these residues were <scene name='PfEBA-140/Asp247/1'>Asp247</scene> and <scene name='PfEBA-140/Arg254/1'>Arg254</scene>. In F2, these residues were <scene name='PfEBA-140/Arg485/1'>Arg485</scene> and <scene name='PfEBA-140/Asp554/2'>Asp554</scene>. When these four residues were individually mutated to Alanine, erythrocyte binding was heavily interrupted. This can be observed in the figure to the right.<ref name="Lin">PMID: 22989878</ref>[[Image:DecreasedBinding.png | thumb | 100px | alt=text | Mutation of Asp247, Arg254, Arg485, and Asp554]] The decreased erythrocyte binding confirms that these <scene name='PfEBA-140/Four/1'>four residues</scene> found in the basic patch of RII PfEBA-140 are crucial to erythrocyte binding. As a control, residues on the opposite side of RII PfEBA-140 were mutated to Alanine. These residues were Asn394 and Glu605. Mutation of these two residues had no effect on erythrocyte binding.<ref name="Lin">PMID: 22989878</ref>[[Image:FullBinding.png | thumb | 100px | alt=text | Mutation of Asn394 and Glu605]] These results fortify the finding that erythrocyte binding takes place in the basic patch discussed earlier.

 
 
 
 
 

==Structural Differences in PfEBA-140 from other EBL Ligands==
<Structure load='4GF2' size='400' frame='true' align='left' caption='Crystal Structure of PfEBA-140 (PDB entry [[4GF2]])' scene=''/>
There are three unique structural elements that set RII PfEBA-140 apart from other EBL ligands. These structures are the likely reason behind RII PfEBA-140 binding GPC so specifically.
===Disulfide Bonds===
[[Image:ModifiedPattern.png | thumb | 300px | alt=text | Disulfide Bonding Patterns]]
There are twenty-six <scene name='PfEBA-140/Cysteines/1'>cysteines</scene> in RII PfEBA-140, all of which are involved in <scene name='PfEBA-140/Disulfidebonds/1'>disulfide bonding</scene> (disulfide bonds shown in black).<ref name="Lin">PMID: 22989878</ref> In RII PfEBA-140, two modified disulfide patterns were found to be different than in RII PfEBA-175, RII PvDBP, and RII PkDBP. In the latter three RII domains, there is a disulfide linkage between Cys7 and Cys9 and between Cys8 and Cys12. In RII PfEBA-140, however, Cys7 is disulfide bonded to Cys8 and Cys9 is disulfide bonded to Cys12.<ref name="Lin">PMID: 22989878</ref>
===Alpha Helix Structure===
Another difference between RII PfEBA-140 and other EBL ligands can be found in the F2 domain. In EBL ligands of other ''Plasmodium'' species, there are β-fingers with either long or short loops which may or may not be important for receptor binding.<ref name="Lin">PMID: 22989878</ref> While this β-finger is found in F1 of PfEBA-140, it is not found in F2, as it is replaced with an <scene name='PfEBA-140/Alphabeta/1'>α-helix</scene>.<ref name="Lin">PMID: 22989878</ref>
===Lack of Kink in F1===
A third difference between RII PfEBA-140 and other EBL ligands is the lack of a glycine residue in F1. This glycine is present in F1 PfEBA-175, F2 PfEBA-175, PvDBP, PkDBP-α, and F2 PfEBA-140. In F2 PfEBA-140, this glycine is residue 627.<ref name="Lin">PMID: 22989878</ref> In these EBL ligands create a kink due to glycine's flexibility. If this glycine residue were present in F1 of PfEBA-140, it would result in a kink in subdomain 3.<ref name="Lin">PMID: 22989878</ref> This kink would create a hinge angle between the two DBL domains that would allow for dimerization of PfEBA-140 upon erythrocyte binding. For this reason, PfEBA-140 is believed to engage erythrocytes as a monomer, unlike other EBL ligands, which bind erythrocytes as a dimer.
 
 
These three structural differences are believed to be responsible for the specificity of RII PfEBA-140.

==References==
<references />

Malaria Parasite Plasmodium falciparum Erythrocyte Binding Antigen 140

2012-11-15T23:40:00Z

Emily Lum: /* Basic Patch */

== Introduction ==
In 2010, malaria caused over 650,000 deaths.<ref name="World Health Organization">http://www.who.int/mediacentre/factsheets/fs094/en/index.html</ref> While this disease is both preventable and curable, many of those that live in areas where the disease is endemic do not have access to such resources. Although there is a vaccine currently undergoing clinical trials, results are not expected until 2014.<ref name="World Health Organization">http://www.who.int/mediacentre/factsheets/fs094/en/index.html</ref> The most recent vaccine research has focused on understanding a group of proteins in the erythrocyte-binding ligand (EBL) family. These proteins, which are found in the ''Plasmodium'' species, facilitate entry into erythrocytes during malarial infection by creating tight junctions between the host erythrocytes and parasite membranes. <ref name="Lin">PMID: 22989878</ref> There are four family members: erythrocyte-binding antigen 175 (PfEBA-175), erythrocyte-binding ligand 1 (PfEBL-1), erythrocyte-binding antigen 140 (PfEBA-140), and erythrocyte-binding antigen 181 (PfEBA-181).<ref name="Lin">PMID: 22989878</ref> PfEBA-140 binds glycophorin C on host erythrocytes,<ref name="Lin">PMID: 22989878</ref> which helps maintain erythrocyte shape and regulates membrane material properties.<ref name="Glycophorin C">http://en.wikipedia.org/wiki/Glycophorin_C</ref> Understanding the mechanism by which PfEBA-140 recognizes and engages glycophorin C on erythrocytes may lead to the future development of a new malaria vaccine, as it has been found to be immunogenic.<ref name="Ford">PMID: 17984363</ref>

==General Structure==
<Structure load='4GF2' size='400' frame='true' align='left' caption='Crystal Structure of PfEBA-140 (PDB entry [[4GF2]])' scene=''/>
[[Image:SubdomainsF1F2.png | thumb | 300px | alt=text | Subdomains of F1 and F2]]
[[Image:IndividualSubdomains.png | thumb | 300px | alt=text | Subdomains of F1 and F2]]
The EBL family members, including PfEBA-140, are made up of two regions, region II (RII) (shown to the right) and region VI.<ref name="Lin">PMID: 22989878</ref> Region II is responsible for receptor binding in all EBL family members.<ref name="Lin">PMID: 22989878</ref> RII is composed of <scene name='PfEBA-140/Rii/1'>two Duffy binding-like (DBL) domains</scene>, F1 (purple) and F2 (teal).<ref name="Lin">PMID: 22989878</ref> These two DBL domains are connected by a <scene name='PfEBA-140/Helical_linker/2'>short helical linker</scene>.<ref name="Lin">PMID: 22989878</ref> The DBL protein fold is unique to the ''Plasmodium'' species. Not only does it have the ability to recognize and bind many erythrocyte cell receptors, but it also mediates microvasculature adherence of infected erythrocytes by erythrocyte membrane protein 1 (PfEMP1).<ref name="Lin">PMID: 22989878</ref> Each DBL domain is composed of three subdomains, illustrated in the pictures to the right.

In the top image, the subdomains S1, S2, and S3 of each of the F1 and F2 domains, as well as the helical linker, are illustrated.<ref name="Lin">PMID: 22989878</ref>
In the bottom image, structures of the individual subdomains are illustrated.<ref name="Lin">PMID: 22989878</ref>

The colors are the same in both images: F1 subdomain 1 is shown in bronze, subdomain 2 in orange, subdomain 3 in dark orange; F2 subdomain 1 is shown in dark blue, subdomain 2 in blue, and subdomain 3 in light blue.

==Binding of RII PfEBA-140==
<Structure load='4GF2' size='400' frame='true' align='left' caption='Crystal Structure of PfEBA-140 (PDB entry [[4GF2]])' scene=''/>
===Requirement of Both DBL Domains===
[[Image:Binding.png | thumb | 100px | alt=text | Erythrocyte Binding of Full Length and Individual RII Domains]]Since the DBL domains of RII are highly conserved, the ability of the individual DBL domains to bind erythrocytes was tested. To do this, constructs containing the full-length RII PfEBA-140 and each individual DBL domain were tested using a rosetting assay. Both brightfield microscopy and green fluorescence protein (GFP) were used to visualize erythrocyte binding. In the upper panel, a construct expressing only GFP was used as a control. The lower panel of Figure A illustrates the extensive erythrocyte binding of the full length RII construct. In Figure B, the lack of black dots in the brightfield microscopy and the smaller quantity of green fluorescence illustrates that F1 and F2 are unable to independently bind erythrocytes. This result suggests that both domains equally participate in engaging erythrocytes.<ref name="Lin">PMID: 22989878</ref>
===Basic Patch===
[[Image:Basic Patch.png | thumb | 100px | alt=text | Erythrocyte Binding of Full Length and Individual RII Domains]]
Since the two DBL domains bind acidic GPC, there must be a number of basic charges that interact with the acidic GPC residues. This idea is supported by visualizing the <scene name='PfEBA-140/Basic_patch/1'>positive and negative residues</scene> found in RII PfEBA-140. The positive side chain residues are red while the negative side chain residues are blue. On the side shown, there are a number of residues with positive side chains, creating a basic patch. This basic patch, which forms an arch spanning the two DBL domains, is thought to interact with the acidic GPC. It is further believed that this is where RII PfEBA-140 binds GPC because if the molecule is flipped 180°, there is no concentration of positive charges. The basic patch may be better visualized in the picture to the right.<ref name="Lin">PMID: 22989878</ref>
To test whether this basic patch was indeed responsible for erythrocyte binding, two residues in each DBL domain were chosen and mutated to Alanine. In F1, these residues were <scene name='PfEBA-140/Asp247/1'>Asp247</scene> and <scene name='PfEBA-140/Arg254/1'>Arg254</scene>. In F2, these residues were <scene name='PfEBA-140/Arg485/1'>Arg485</scene> and <scene name='PfEBA-140/Asp554/2'>Asp554</scene>. When these four residues were individually mutated to Alanine, erythrocyte binding was heavily interrupted. This can be observed in the figure to the right.<ref name="Lin">PMID: 22989878</ref>[[Image:DecreasedBinding.png | thumb | 100px | alt=text | Mutation of Asp247, Arg254, Arg485, and Asp554]] The decreased erythrocyte binding confirms that these <scene name='PfEBA-140/Four/1'>four residues</scene> found in the basic patch of RII PfEBA-140 are crucial to erythrocyte binding. As a control, residues on the opposite side of RII PfEBA-140 were mutated to Alanine. These residues were Asn394 and Glu605. Mutation of these two residues had no effect on erythrocyte binding.<ref name="Lin">PMID: 22989878</ref>[[Image:FullBinding.png | thumb | 100px | alt=text | Mutation of Asn394 and Glu605]] These results fortify the finding that erythrocyte binding takes place in the basic patch discussed earlier.

 
 
 
 
 

==Structural Differences in PfEBA-140 from other EBL Ligands==
<Structure load='4GF2' size='400' frame='true' align='left' caption='Crystal Structure of PfEBA-140 (PDB entry [[4GF2]])' scene=''/>
There are three unique structural elements that set RII PfEBA-140 apart from other EBL ligands. These structures are the likely reason behind RII PfEBA-140 binding GPC so specifically.
===Disulfide Bonds===
[[Image:ModifiedPattern.png | thumb | 300px | alt=text | Disulfide Bonding Patterns]]
There are twenty-six <scene name='PfEBA-140/Cysteines/1'>cysteines</scene> in RII PfEBA-140, all of which are involved in <scene name='PfEBA-140/Disulfidebonds/1'>disulfide bonding</scene> (disulfide bonds shown in black).<ref name="Lin">PMID: 22989878</ref> In RII PfEBA-140, two modified disulfide patterns were found to be different than in RII PfEBA-175, RII PvDBP, and RII PkDBP. In the latter three RII domains, there is a disulfide linkage between Cys7 and Cys9 and between Cys8 and Cys12. In RII PfEBA-140, however, Cys7 is disulfide bonded to Cys8 and Cys9 is disulfide bonded to Cys12.<ref name="Lin">PMID: 22989878</ref>
===Alpha Helix Structure===
Another difference between RII PfEBA-140 and other EBL ligands can be found in the F2 domain. In EBL ligands of other ''Plasmodium'' species, there are β-fingers with either long or short loops which may or may not be important for receptor binding.<ref name="Lin">PMID: 22989878</ref> While this β-finger is found in F1 of PfEBA-140, it is not found in F2, as it is replaced with an <scene name='PfEBA-140/Alphabeta/1'>α-helix</scene>.<ref name="Lin">PMID: 22989878</ref>
===Lack of Kink in F1===
A third difference between RII PfEBA-140 and other EBL ligands is the lack of a glycine residue in F1. This glycine is present in F1 PfEBA-175, F2 PfEBA-175, PvDBP, PkDBP-α, and F2 PfEBA-140. In F2 PfEBA-140, this glycine is residue 627.<ref name="Lin">PMID: 22989878</ref> In these EBL ligands create a kink due to glycine's flexibility. If this glycine residue were present in F1 of PfEBA-140, it would result in a kink in subdomain 3.<ref name="Lin">PMID: 22989878</ref> This kink would create a hinge angle between the two DBL domains that would allow for dimerization of PfEBA-140 upon erythrocyte binding. For this reason, PfEBA-140 is believed to engage erythrocytes as a monomer, unlike other EBL ligands, which bind erythrocytes as a dimer.
 
 
These three structural differences are believed to be responsible for the specificity of RII PfEBA-140.

==References==
<references />

Malaria Parasite Plasmodium falciparum Erythrocyte Binding Antigen 140

2012-11-15T23:32:45Z

Emily Lum: /* Introduction */

== Introduction ==
In 2010, malaria caused over 650,000 deaths.<ref name="World Health Organization">http://www.who.int/mediacentre/factsheets/fs094/en/index.html</ref> While this disease is both preventable and curable, many of those that live in areas where the disease is endemic do not have access to such resources. Although there is a vaccine currently undergoing clinical trials, results are not expected until 2014.<ref name="World Health Organization">http://www.who.int/mediacentre/factsheets/fs094/en/index.html</ref> The most recent vaccine research has focused on understanding a group of proteins in the erythrocyte-binding ligand (EBL) family. These proteins, which are found in the ''Plasmodium'' species, facilitate entry into erythrocytes during malarial infection by creating tight junctions between the host erythrocytes and parasite membranes. <ref name="Lin">PMID: 22989878</ref> There are four family members: erythrocyte-binding antigen 175 (PfEBA-175), erythrocyte-binding ligand 1 (PfEBL-1), erythrocyte-binding antigen 140 (PfEBA-140), and erythrocyte-binding antigen 181 (PfEBA-181).<ref name="Lin">PMID: 22989878</ref> PfEBA-140 binds glycophorin C on host erythrocytes,<ref name="Lin">PMID: 22989878</ref> which helps maintain erythrocyte shape and regulates membrane material properties.<ref name="Glycophorin C">http://en.wikipedia.org/wiki/Glycophorin_C</ref> Understanding the mechanism by which PfEBA-140 recognizes and engages glycophorin C on erythrocytes may lead to the future development of a new malaria vaccine, as it has been found to be immunogenic.<ref name="Ford">PMID: 17984363</ref>

==General Structure==
<Structure load='4GF2' size='400' frame='true' align='left' caption='Crystal Structure of PfEBA-140 (PDB entry [[4GF2]])' scene=''/>
[[Image:SubdomainsF1F2.png | thumb | 300px | alt=text | Subdomains of F1 and F2]]
[[Image:IndividualSubdomains.png | thumb | 300px | alt=text | Subdomains of F1 and F2]]
The EBL family members, including PfEBA-140, are made up of two regions, region II (RII) (shown to the right) and region VI.<ref name="Lin">PMID: 22989878</ref> Region II is responsible for receptor binding in all EBL family members.<ref name="Lin">PMID: 22989878</ref> RII is composed of <scene name='PfEBA-140/Rii/1'>two Duffy binding-like (DBL) domains</scene>, F1 (purple) and F2 (teal).<ref name="Lin">PMID: 22989878</ref> These two DBL domains are connected by a <scene name='PfEBA-140/Helical_linker/2'>short helical linker</scene>.<ref name="Lin">PMID: 22989878</ref> The DBL protein fold is unique to the ''Plasmodium'' species. Not only does it have the ability to recognize and bind many erythrocyte cell receptors, but it also mediates microvasculature adherence of infected erythrocytes by erythrocyte membrane protein 1 (PfEMP1).<ref name="Lin">PMID: 22989878</ref> Each DBL domain is composed of three subdomains, illustrated in the pictures to the right.

In the top image, the subdomains S1, S2, and S3 of each of the F1 and F2 domains, as well as the helical linker, are illustrated.<ref name="Lin">PMID: 22989878</ref>
In the bottom image, structures of the individual subdomains are illustrated.<ref name="Lin">PMID: 22989878</ref>

The colors are the same in both images: F1 subdomain 1 is shown in bronze, subdomain 2 in orange, subdomain 3 in dark orange; F2 subdomain 1 is shown in dark blue, subdomain 2 in blue, and subdomain 3 in light blue.

==Binding of RII PfEBA-140==
<Structure load='4GF2' size='400' frame='true' align='left' caption='Crystal Structure of PfEBA-140 (PDB entry [[4GF2]])' scene=''/>
===Requirement of Both DBL Domains===
[[Image:Binding.png | thumb | 100px | alt=text | Erythrocyte Binding of Full Length and Individual RII Domains]]Since the DBL domains of RII are highly conserved, the ability of the individual DBL domains to bind erythrocytes was tested. To do this, constructs containing the full-length RII PfEBA-140 and each individual DBL domain were tested using a rosetting assay. Both brightfield microscopy and green fluorescence protein (GFP) were used to visualize erythrocyte binding. In the upper panel, a construct expressing only GFP was used as a control. The lower panel of Figure A illustrates the extensive erythrocyte binding of the full length RII construct. In Figure B, the lack of black dots in the brightfield microscopy and the smaller quantity of green fluorescence illustrates that F1 and F2 are unable to independently bind erythrocytes. This result suggests that both domains equally participate in engaging erythrocytes.<ref name="Lin">PMID: 22989878</ref>
===Basic Patch===
[[Image:Basic Patch.png | thumb | 100px | alt=text | Erythrocyte Binding of Full Length and Individual RII Domains]]
Since the two DBL domains bind acidic GPC, there must be a number of basic charges that interact with the acidic GPC residues. This idea is supported by visualizing the <scene name='PfEBA-140/Basic_patch/1'>positive and negative residues</scene> found in RII PfEBA-140. The positive side chain residues are red while the negative side chain residues are blue. On the side shown, there are a number of residues with positive side chains, creating a basic patch. This basic patch, which forms an arch spanning the two DBL domains. It is thought that this basic arch interacts with the acidic GPC. It is further believed that this is where RII PfEBA-140 binds GPC because if the molecule is flipped 180°, there is no concentration of positive charges. The basic patch may be better visualized in the picture to the right.<ref name="Lin">PMID: 22989878</ref>
To test whether this basic patch was indeed responsible for erythrocyte binding, two residues in each DBL domain were chosen and mutated to Alanine. In F1, these residues were <scene name='PfEBA-140/Asp247/1'>Asp247</scene> and <scene name='PfEBA-140/Arg254/1'>Arg254</scene>. In F2, these residues were <scene name='PfEBA-140/Arg485/1'>Arg485</scene> and <scene name='PfEBA-140/Asp554/2'>Asp554</scene>. When these four residues were individually mutated to Alanine, erythrocyte binding was heavily interrupted. This can be observed in the figure to the right.<ref name="Lin">PMID: 22989878</ref>[[Image:DecreasedBinding.png | thumb | 100px | alt=text | Mutation of Asp247, Arg254, Arg485, and Asp554]] The decreased erythrocyte binding confirms that these <scene name='PfEBA-140/Four/1'>four residues</scene> found in the basic patch of RII PfEBA-140 are crucial to erythrocyte binding. As a control, residues on the opposite side of RII PfEBA-140 were mutated to Alanine. These residues were Asn394 and Glu605. Mutation of these two residues had no effect on erythrocyte binding.<ref name="Lin">PMID: 22989878</ref>[[Image:FullBinding.png | thumb | 100px | alt=text | Mutation of Asn394 and Glu605]] These results fortify the finding that erythrocyte binding takes place in the basic patch discussed earlier.

 
 
 
 
 

==Structural Differences in PfEBA-140 from other EBL Ligands==
<Structure load='4GF2' size='400' frame='true' align='left' caption='Crystal Structure of PfEBA-140 (PDB entry [[4GF2]])' scene=''/>
There are three unique structural elements that set RII PfEBA-140 apart from other EBL ligands. These structures are the likely reason behind RII PfEBA-140 binding GPC so specifically.
===Disulfide Bonds===
[[Image:ModifiedPattern.png | thumb | 300px | alt=text | Disulfide Bonding Patterns]]
There are twenty-six <scene name='PfEBA-140/Cysteines/1'>cysteines</scene> in RII PfEBA-140, all of which are involved in <scene name='PfEBA-140/Disulfidebonds/1'>disulfide bonding</scene> (disulfide bonds shown in black).<ref name="Lin">PMID: 22989878</ref> In RII PfEBA-140, two modified disulfide patterns were found to be different than in RII PfEBA-175, RII PvDBP, and RII PkDBP. In the latter three RII domains, there is a disulfide linkage between Cys7 and Cys9 and between Cys8 and Cys12. In RII PfEBA-140, however, Cys7 is disulfide bonded to Cys8 and Cys9 is disulfide bonded to Cys12.<ref name="Lin">PMID: 22989878</ref>
===Alpha Helix Structure===
Another difference between RII PfEBA-140 and other EBL ligands can be found in the F2 domain. In EBL ligands of other ''Plasmodium'' species, there are β-fingers with either long or short loops which may or may not be important for receptor binding.<ref name="Lin">PMID: 22989878</ref> While this β-finger is found in F1 of PfEBA-140, it is not found in F2, as it is replaced with an <scene name='PfEBA-140/Alphabeta/1'>α-helix</scene>.<ref name="Lin">PMID: 22989878</ref>
===Lack of Kink in F1===
A third difference between RII PfEBA-140 and other EBL ligands is the lack of a glycine residue in F1. This glycine is present in F1 PfEBA-175, F2 PfEBA-175, PvDBP, PkDBP-α, and F2 PfEBA-140. In F2 PfEBA-140, this glycine is residue 627.<ref name="Lin">PMID: 22989878</ref> In these EBL ligands create a kink due to glycine's flexibility. If this glycine residue were present in F1 of PfEBA-140, it would result in a kink in subdomain 3.<ref name="Lin">PMID: 22989878</ref> This kink would create a hinge angle between the two DBL domains that would allow for dimerization of PfEBA-140 upon erythrocyte binding. For this reason, PfEBA-140 is believed to engage erythrocytes as a monomer, unlike other EBL ligands, which bind erythrocytes as a dimer.
 
 
These three structural differences are believed to be responsible for the specificity of RII PfEBA-140.

==References==
<references />

Malaria Parasite Plasmodium falciparum Erythrocyte Binding Antigen 140

2012-11-15T22:52:11Z

Emily Lum:

== Introduction ==
In 2010, malaria caused over 650,000 deaths.<ref name="World Health Organization">http://www.who.int/mediacentre/factsheets/fs094/en/index.html</ref> While this disease is both preventable and curable, many of those that live in areas where the disease is endemic do not have access to such resources. Although there is a vaccine currently undergoing clinical trials, results are not expected until 2014.<ref name="World Health Organization">http://www.who.int/mediacentre/factsheets/fs094/en/index.html</ref> The most recent vaccine research has focused on understanding a group of proteins in the erythrocyte-binding ligand (EBL) family. These proteins, which are found in the ''Plasmodium'' species, facilitate entry into erythrocytes during malarial infection by creating tight junctions between the host erythrocytes and parasite membranes. <ref name="Lin">PMID: 22989878</ref> There are four family members: erythrocyte-binding antigen 175 (PfEBA-175), erythrocyte-binding ligand 1 (PfEBL-1), erythrocyte-binding antigen 140 (PfEBA-140), and erythrocyte-binding antigen 181 (PfEBA-181).<ref name="Lin">PMID: 22989878</ref> PfEBA-140 binds glycophorin C on host erythrocytes,<ref name="Lin">PMID: 22989878</ref> which helps maintain erythrocyte shape and regulates membrane material properties.<ref name="Glycophorin C">http://en.wikipedia.org/wiki/Glycophorin_C</ref> Understanding the mechanism by which PfEBA-140 recognizes and engages glycophorin C on erythrocytes may lead to the future development of a new malaria vaccine, because it has been found to be immunogenic.<ref name="Ford">PMID: 17984363</ref>

==General Structure==
<Structure load='4GF2' size='400' frame='true' align='left' caption='Crystal Structure of PfEBA-140 (PDB entry [[4GF2]])' scene=''/>
[[Image:SubdomainsF1F2.png | thumb | 300px | alt=text | Subdomains of F1 and F2]]
[[Image:IndividualSubdomains.png | thumb | 300px | alt=text | Subdomains of F1 and F2]]
The EBL family members, including PfEBA-140, are made up of two regions, region II (RII) (shown to the right) and region VI.<ref name="Lin">PMID: 22989878</ref> Region II is responsible for receptor binding in all EBL family members.<ref name="Lin">PMID: 22989878</ref> RII is composed of <scene name='PfEBA-140/Rii/1'>two Duffy binding-like (DBL) domains</scene>, F1 (purple) and F2 (teal).<ref name="Lin">PMID: 22989878</ref> These two DBL domains are connected by a <scene name='PfEBA-140/Helical_linker/2'>short helical linker</scene>.<ref name="Lin">PMID: 22989878</ref> The DBL protein fold is unique to the ''Plasmodium'' species. Not only does it have the ability to recognize and bind many erythrocyte cell receptors, but it also mediates microvasculature adherence of infected erythrocytes by erythrocyte membrane protein 1 (PfEMP1).<ref name="Lin">PMID: 22989878</ref> Each DBL domain is composed of three subdomains, illustrated in the pictures to the right.

In the top image, the subdomains S1, S2, and S3 of each of the F1 and F2 domains, as well as the helical linker, are illustrated.<ref name="Lin">PMID: 22989878</ref>
In the bottom image, structures of the individual subdomains are illustrated.<ref name="Lin">PMID: 22989878</ref>

The colors are the same in both images: F1 subdomain 1 is shown in bronze, subdomain 2 in orange, subdomain 3 in dark orange; F2 subdomain 1 is shown in dark blue, subdomain 2 in blue, and subdomain 3 in light blue.

==Binding of RII PfEBA-140==
<Structure load='4GF2' size='400' frame='true' align='left' caption='Crystal Structure of PfEBA-140 (PDB entry [[4GF2]])' scene=''/>
===Requirement of Both DBL Domains===
[[Image:Binding.png | thumb | 100px | alt=text | Erythrocyte Binding of Full Length and Individual RII Domains]]Since the DBL domains of RII are highly conserved, the ability of the individual DBL domains to bind erythrocytes was tested. To do this, constructs containing the full-length RII PfEBA-140 and each individual DBL domain were tested using a rosetting assay. Both brightfield microscopy and green fluorescence protein (GFP) were used to visualize erythrocyte binding. In the upper panel, a construct expressing only GFP was used as a control. The lower panel of Figure A illustrates the extensive erythrocyte binding of the full length RII construct. In Figure B, the lack of black dots in the brightfield microscopy and the smaller quantity of green fluorescence illustrates that F1 and F2 are unable to independently bind erythrocytes. This result suggests that both domains equally participate in engaging erythrocytes.<ref name="Lin">PMID: 22989878</ref>
===Basic Patch===
[[Image:Basic Patch.png | thumb | 100px | alt=text | Erythrocyte Binding of Full Length and Individual RII Domains]]
Since the two DBL domains bind acidic GPC, there must be a number of basic charges that interact with the acidic GPC residues. This idea is supported by visualizing the <scene name='PfEBA-140/Basic_patch/1'>positive and negative residues</scene> found in RII PfEBA-140. The positive side chain residues are red while the negative side chain residues are blue. On the side shown, there are a number of residues with positive side chains, creating a basic patch. This basic patch, which forms an arch spanning the two DBL domains. It is thought that this basic arch interacts with the acidic GPC. It is further believed that this is where RII PfEBA-140 binds GPC because if the molecule is flipped 180°, there is no concentration of positive charges. The basic patch may be better visualized in the picture to the right.<ref name="Lin">PMID: 22989878</ref>
To test whether this basic patch was indeed responsible for erythrocyte binding, two residues in each DBL domain were chosen and mutated to Alanine. In F1, these residues were <scene name='PfEBA-140/Asp247/1'>Asp247</scene> and <scene name='PfEBA-140/Arg254/1'>Arg254</scene>. In F2, these residues were <scene name='PfEBA-140/Arg485/1'>Arg485</scene> and <scene name='PfEBA-140/Asp554/2'>Asp554</scene>. When these four residues were individually mutated to Alanine, erythrocyte binding was heavily interrupted. This can be observed in the figure to the right.<ref name="Lin">PMID: 22989878</ref>[[Image:DecreasedBinding.png | thumb | 100px | alt=text | Mutation of Asp247, Arg254, Arg485, and Asp554]] The decreased erythrocyte binding confirms that these <scene name='PfEBA-140/Four/1'>four residues</scene> found in the basic patch of RII PfEBA-140 are crucial to erythrocyte binding. As a control, residues on the opposite side of RII PfEBA-140 were mutated to Alanine. These residues were Asn394 and Glu605. Mutation of these two residues had no effect on erythrocyte binding.<ref name="Lin">PMID: 22989878</ref>[[Image:FullBinding.png | thumb | 100px | alt=text | Mutation of Asn394 and Glu605]] These results fortify the finding that erythrocyte binding takes place in the basic patch discussed earlier.

 
 
 
 
 

==Structural Differences in PfEBA-140 from other EBL Ligands==
<Structure load='4GF2' size='400' frame='true' align='left' caption='Crystal Structure of PfEBA-140 (PDB entry [[4GF2]])' scene=''/>
There are three unique structural elements that set RII PfEBA-140 apart from other EBL ligands. These structures are the likely reason behind RII PfEBA-140 binding GPC so specifically.
===Disulfide Bonds===
[[Image:ModifiedPattern.png | thumb | 300px | alt=text | Disulfide Bonding Patterns]]
There are twenty-six <scene name='PfEBA-140/Cysteines/1'>cysteines</scene> in RII PfEBA-140, all of which are involved in <scene name='PfEBA-140/Disulfidebonds/1'>disulfide bonding</scene> (disulfide bonds shown in black).<ref name="Lin">PMID: 22989878</ref> In RII PfEBA-140, two modified disulfide patterns were found to be different than in RII PfEBA-175, RII PvDBP, and RII PkDBP. In the latter three RII domains, there is a disulfide linkage between Cys7 and Cys9 and between Cys8 and Cys12. In RII PfEBA-140, however, Cys7 is disulfide bonded to Cys8 and Cys9 is disulfide bonded to Cys12.<ref name="Lin">PMID: 22989878</ref>
===Alpha Helix Structure===
Another difference between RII PfEBA-140 and other EBL ligands can be found in the F2 domain. In EBL ligands of other ''Plasmodium'' species, there are β-fingers with either long or short loops which may or may not be important for receptor binding.<ref name="Lin">PMID: 22989878</ref> While this β-finger is found in F1 of PfEBA-140, it is not found in F2, as it is replaced with an <scene name='PfEBA-140/Alphabeta/1'>α-helix</scene>.<ref name="Lin">PMID: 22989878</ref>
===Lack of Kink in F1===
A third difference between RII PfEBA-140 and other EBL ligands is the lack of a glycine residue in F1. This glycine is present in F1 PfEBA-175, F2 PfEBA-175, PvDBP, PkDBP-α, and F2 PfEBA-140. In F2 PfEBA-140, this glycine is residue 627.<ref name="Lin">PMID: 22989878</ref> In these EBL ligands create a kink due to glycine's flexibility. If this glycine residue were present in F1 of PfEBA-140, it would result in a kink in subdomain 3.<ref name="Lin">PMID: 22989878</ref> This kink would create a hinge angle between the two DBL domains that would allow for dimerization of PfEBA-140 upon erythrocyte binding. For this reason, PfEBA-140 is believed to engage erythrocytes as a monomer, unlike other EBL ligands, which bind erythrocytes as a dimer.
 
 
These three structural differences are believed to be responsible for the specificity of RII PfEBA-140.

==References==
<references />

Malaria Parasite Plasmodium falciparum Erythrocyte Binding Antigen 140

2012-11-15T21:25:10Z

Emily Lum: /* General Structure */

== Introduction ==
In 2010, malaria caused over 650,000 deaths.<ref name="World Health Organization">http://www.who.int/mediacentre/factsheets/fs094/en/index.html</ref> While this disease is both preventable and curable, many of those that live in areas where the disease is endemic do not have access to such resources. Although there is a vaccine currently undergoing clinical trials, results are not expected until 2014.<ref name="World Health Organization">http://www.who.int/mediacentre/factsheets/fs094/en/index.html</ref> The most recent vaccine research has focused on understanding a group of proteins in the erythrocyte-binding ligand (EBL) family. These proteins, which are found in the ''Plasmodium'' species, facilitate entry into erythrocytes during malarial infection by creating tight junctions between the host erythrocytes and parasite membranes. <ref name="Lin">PMID: 22989878</ref> There are four family members: erythrocyte-binding antigen 175 (PfEBA-175), erythrocyte-binding ligand 1 (PfEBL-1), erythrocyte-binding antigen 140 (PfEBA-140), and erythrocyte-binding antigen 181 (PfEBA-181).<ref name="Lin">PMID: 22989878</ref> PfEBA-140 binds glycophorin C on host erythrocytes,<ref name="Lin">PMID: 22989878</ref> which helps maintain erythrocyte shape and regulates membrane material properties.<ref name="Glycophorin C">http://en.wikipedia.org/wiki/Glycophorin_C</ref> Understanding the mechanism by which PfEBA-140 recognizes and engages glycophorin C on erythrocytes may lead to the future development of a new malaria vaccine.

==General Structure==
<Structure load='4GF2' size='400' frame='true' align='left' caption='Crystal Structure of PfEBA-140 (PDB entry [[4GF2]])' scene=''/>
[[Image:SubdomainsF1F2.png | thumb | 300px | alt=text | Subdomains of F1 and F2]]
[[Image:IndividualSubdomains.png | thumb | 300px | alt=text | Subdomains of F1 and F2]]
The EBL family members, including PfEBA-140, are made up of two regions, region II (RII) (shown to the right) and region VI.<ref name="Lin">PMID: 22989878</ref> Region II is responsible for receptor binding in all EBL family members.<ref name="Lin">PMID: 22989878</ref> RII is composed of <scene name='PfEBA-140/Rii/1'>two Duffy binding-like (DBL) domains</scene>, F1 (purple) and F2 (teal).<ref name="Lin">PMID: 22989878</ref> These two DBL domains are connected by a <scene name='PfEBA-140/Helical_linker/2'>short helical linker</scene>.<ref name="Lin">PMID: 22989878</ref> The DBL protein fold is unique to the ''Plasmodium'' species. Not only does it have the ability to recognize and bind many erythrocyte cell receptors, but it also mediates microvasculature adherence of infected erythrocytes by erythrocyte membrane protein 1 (PfEMP1).<ref name="Lin">PMID: 22989878</ref> Each DBL domain is composed of three subdomains, illustrated in the pictures to the right.

In the top image, the subdomains S1, S2, and S3 of each of the F1 and F2 domains, as well as the helical linker, are illustrated.<ref name="Lin">PMID: 22989878</ref>
In the bottom image, structures of the individual subdomains are illustrated.<ref name="Lin">PMID: 22989878</ref>

The colors are the same in both images: F1 subdomain 1 is shown in bronze, subdomain 2 in orange, subdomain 3 in dark orange; F2 subdomain 1 is shown in dark blue, subdomain 2 in blue, and subdomain 3 in light blue.

==Binding of RII PfEBA-140==
<Structure load='4GF2' size='400' frame='true' align='left' caption='Crystal Structure of PfEBA-140 (PDB entry [[4GF2]])' scene=''/>
===Requirement of Both DBL Domains===
[[Image:Binding.png | thumb | 100px | alt=text | Erythrocyte Binding of Full Length and Individual RII Domains]]Since the DBL domains of RII are highly conserved, the ability of the individual DBL domains to bind erythrocytes was tested. To do this, constructs containing the full-length RII PfEBA-140 and each individual DBL domain were tested using a rosetting assay. Both brightfield microscopy and green fluorescence protein (GFP) were used to visualize erythrocyte binding. In the upper panel, a construct expressing only GFP was used as a control. The lower panel of Figure A illustrates the extensive erythrocyte binding of the full length RII construct. In Figure B, the lack of black dots in the brightfield microscopy and the smaller quantity of green fluorescence illustrates that F1 and F2 are unable to independently bind erythrocytes. This result suggests that both domains equally participate in engaging erythrocytes.<ref name="Lin">PMID: 22989878</ref>
===Basic Patch===
[[Image:Basic Patch.png | thumb | 100px | alt=text | Erythrocyte Binding of Full Length and Individual RII Domains]]
Since the two DBL domains bind acidic GPC, there must be a number of basic charges that interact with the acidic GPC residues. This idea is supported by visualizing the <scene name='PfEBA-140/Basic_patch/1'>positive and negative residues</scene> found in RII PfEBA-140. The positive side chain residues are red while the negative side chain residues are blue. On the side shown, there are a number of residues with positive side chains, creating a basic patch. This basic patch, which forms an arch spanning the two DBL domains. It is thought that this basic arch interacts with the acidic GPC. It is further believed that this is where RII PfEBA-140 binds GPC because if the molecule is flipped 180°, there is no concentration of positive charges. The basic patch may be better visualized in the picture to the right.<ref name="Lin">PMID: 22989878</ref>
To test whether this basic patch was indeed responsible for erythrocyte binding, two residues in each DBL domain were chosen and mutated to Alanine. In F1, these residues were <scene name='PfEBA-140/Asp247/1'>Asp247</scene> and <scene name='PfEBA-140/Arg254/1'>Arg254</scene>. In F2, these residues were <scene name='PfEBA-140/Arg485/1'>Arg485</scene> and <scene name='PfEBA-140/Asp554/2'>Asp554</scene>. When these four residues were individually mutated to Alanine, erythrocyte binding was heavily interrupted. This can be observed in the figure to the right.<ref name="Lin">PMID: 22989878</ref>[[Image:DecreasedBinding.png | thumb | 100px | alt=text | Mutation of Asp247, Arg254, Arg485, and Asp554]] The decreased erythrocyte binding confirms that these <scene name='PfEBA-140/Four/1'>four residues</scene> found in the basic patch of RII PfEBA-140 are crucial to erythrocyte binding. As a control, residues on the opposite side of RII PfEBA-140 were mutated to Alanine. These residues were Asn394 and Glu605. Mutation of these two residues had no effect on erythrocyte binding.<ref name="Lin">PMID: 22989878</ref>[[Image:FullBinding.png | thumb | 100px | alt=text | Mutation of Asn394 and Glu605]] These results fortify the finding that erythrocyte binding takes place in the basic patch discussed earlier.

 
 
 
 
 

==Structural Differences in PfEBA-140 from other EBL Ligands==
<Structure load='4GF2' size='400' frame='true' align='left' caption='Crystal Structure of PfEBA-140 (PDB entry [[4GF2]])' scene=''/>
There are three unique structural elements that set RII PfEBA-140 apart from other EBL ligands. These structures are the likely reason behind RII PfEBA-140 binding GPC so specifically.
===Disulfide Bonds===
[[Image:ModifiedPattern.png | thumb | 300px | alt=text | Disulfide Bonding Patterns]]
There are twenty-six <scene name='PfEBA-140/Cysteines/1'>cysteines</scene> in RII PfEBA-140, all of which are involved in <scene name='PfEBA-140/Disulfidebonds/1'>disulfide bonding</scene> (disulfide bonds shown in black).<ref name="Lin">PMID: 22989878</ref> In RII PfEBA-140, two modified disulfide patterns were found to be different than in RII PfEBA-175, RII PvDBP, and RII PkDBP. In the latter three RII domains, there is a disulfide linkage between Cys7 and Cys9 and between Cys8 and Cys12. In RII PfEBA-140, however, Cys7 is disulfide bonded to Cys8 and Cys9 is disulfide bonded to Cys12.<ref name="Lin">PMID: 22989878</ref>
===Alpha Helix Structure===
Another difference between RII PfEBA-140 and other EBL ligands can be found in the F2 domain. In EBL ligands of other ''Plasmodium'' species, there are β-fingers with either long or short loops which may or may not be important for receptor binding.<ref name="Lin">PMID: 22989878</ref> While this β-finger is found in F1 of PfEBA-140, it is not found in F2, as it is replaced with an <scene name='PfEBA-140/Alphabeta/1'>α-helix</scene>.<ref name="Lin">PMID: 22989878</ref>
===Lack of Kink in F1===
A third difference between RII PfEBA-140 and other EBL ligands is the lack of a glycine residue in F1. This glycine is present in F1 PfEBA-175, F2 PfEBA-175, PvDBP, PkDBP-α, and F2 PfEBA-140. In F2 PfEBA-140, this glycine is residue 627.<ref name="Lin">PMID: 22989878</ref> In these EBL ligands create a kink due to glycine's flexibility. If this glycine residue were present in F1 of PfEBA-140, it would result in a kink in subdomain 3.<ref name="Lin">PMID: 22989878</ref> This kink would create a hinge angle between the two DBL domains that would allow for dimerization of PfEBA-140 upon erythrocyte binding. For this reason, PfEBA-140 is believed to engage erythrocytes as a monomer, unlike other EBL ligands, which bind erythrocytes as a dimer.
 
 
These three structural differences are believed to be responsible for the specificity of RII PfEBA-140.

==Importance to the Future of Malaria==
RII PfEBA-140 binds irreversibly to erythrocytes, creating a tight junction between the ''Plasmodium falciparum'' merozoites and the host red blood cell. Although more research needs to be performed, PfEBA-140 would serve as a good target for future malaria vaccines as it has been found to be immunogenic.<ref name="Ford">PMID: 17984363</ref>
==References==
<references />

PfEBA-140

2012-11-15T21:11:21Z

Emily Lum: PfEBA-140 moved to User:Emily Lum/PfEBA-140: I am doing a class presentation on this page and do not want any of the content changed.

#REDIRECT [[User:Emily Lum/PfEBA-140]]

Malaria Parasite Plasmodium falciparum Erythrocyte Binding Antigen 140

2012-11-15T21:11:21Z

Emily Lum: PfEBA-140 moved to User:Emily Lum/PfEBA-140: I am doing a class presentation on this page and do not want any of the content changed.

== Introduction ==
In 2010, malaria caused over 650,000 deaths.<ref name="World Health Organization">http://www.who.int/mediacentre/factsheets/fs094/en/index.html</ref> While this disease is both preventable and curable, many of those that live in areas where the disease is endemic do not have access to such resources. Although there is a vaccine currently undergoing clinical trials, results are not expected until 2014.<ref name="World Health Organization">http://www.who.int/mediacentre/factsheets/fs094/en/index.html</ref> The most recent vaccine research has focused on understanding a group of proteins in the erythrocyte-binding ligand (EBL) family. These proteins, which are found in the ''Plasmodium'' species, facilitate entry into erythrocytes during malarial infection by creating tight junctions between the host erythrocytes and parasite membranes. <ref name="Lin">PMID: 22989878</ref> There are four family members: erythrocyte-binding antigen 175 (PfEBA-175), erythrocyte-binding ligand 1 (PfEBL-1), erythrocyte-binding antigen 140 (PfEBA-140), and erythrocyte-binding antigen 181 (PfEBA-181).<ref name="Lin">PMID: 22989878</ref> PfEBA-140 binds glycophorin C on host erythrocytes,<ref name="Lin">PMID: 22989878</ref> which helps maintain erythrocyte shape and regulates membrane material properties.<ref name="Glycophorin C">http://en.wikipedia.org/wiki/Glycophorin_C</ref> Understanding the mechanism by which PfEBA-140 recognizes and engages glycophorin C on erythrocytes may lead to the future development of a new malaria vaccine.

==General Structure==
<Structure load='4GF2' size='400' frame='true' align='left' caption='Crystal Structure of PfEBA-140 (PDB entry [[4GF2]])' scene=''/>
[[Image:SubdomainsF1F2.png | thumb | 300px | alt=text | Subdomains of F1 and F2]]
[[Image:IndividualSubdomains.png | thumb | 300px | alt=text | Subdomains of F1 and F2]]
The EBL family members, including PfEBA-140, are made up of two regions, region II (RII) (shown to the right) and region VI.<ref name="Lin">PMID: 22989878</ref> Region II is responsible for receptor binding in all EBL family members.<ref name="Lin">PMID: 22989878</ref> RII is composed of <scene name='PfEBA-140/Rii/1'>two Duffy binding-like (DBL) domains</scene>, F1 (purple) and F2 (teal).<ref name="Lin">PMID: 22989878</ref> These two DBL domains are connected by a <scene name='PfEBA-140/Helical_linker/2'>short helical linker</scene>.<ref name="Lin">PMID: 22989878</ref> The DBL protein fold is unique to the ''Plasmodium'' species. Not only does it have the ability to recognize and bind many erythrocyte cell receptors, but it also mediates microvasculature adherence of infected erythrocytes by erythrocyte membrane protein 1 (PfEMP1).<ref name="Lin">PMID: 22989878</ref> Each DBL domain is composed of three subdomains, illustrated in the pictures below.

In the top image, the subdomains S1, S2, and S3 of each of the F1 and F2 domains, as well as the helical linker, are illustrated.<ref name="Lin">PMID: 22989878</ref>
In the bottom image, structures of the individual subdomains are illustrated.<ref name="Lin">PMID: 22989878</ref>

The colors are the same in both images: F1 subdomain 1 is shown in bronze, subdomain 2 in orange, subdomain 3 in dark orange; F2 subdomain 1 is shown in dark blue, subdomain 2 in blue, and subdomain 3 in light blue.

==Binding of RII PfEBA-140==
<Structure load='4GF2' size='400' frame='true' align='left' caption='Crystal Structure of PfEBA-140 (PDB entry [[4GF2]])' scene=''/>
===Requirement of Both DBL Domains===
[[Image:Binding.png | thumb | 100px | alt=text | Erythrocyte Binding of Full Length and Individual RII Domains]]Since the DBL domains of RII are highly conserved, the ability of the individual DBL domains to bind erythrocytes was tested. To do this, constructs containing the full-length RII PfEBA-140 and each individual DBL domain were tested using a rosetting assay. Both brightfield microscopy and green fluorescence protein (GFP) were used to visualize erythrocyte binding. In the upper panel, a construct expressing only GFP was used as a control. The lower panel of Figure A illustrates the extensive erythrocyte binding of the full length RII construct. In Figure B, the lack of black dots in the brightfield microscopy and the smaller quantity of green fluorescence illustrates that F1 and F2 are unable to independently bind erythrocytes. This result suggests that both domains equally participate in engaging erythrocytes.<ref name="Lin">PMID: 22989878</ref>
===Basic Patch===
[[Image:Basic Patch.png | thumb | 100px | alt=text | Erythrocyte Binding of Full Length and Individual RII Domains]]
Since the two DBL domains bind acidic GPC, there must be a number of basic charges that interact with the acidic GPC residues. This idea is supported by visualizing the <scene name='PfEBA-140/Basic_patch/1'>positive and negative residues</scene> found in RII PfEBA-140. The positive side chain residues are red while the negative side chain residues are blue. On the side shown, there are a number of residues with positive side chains, creating a basic patch. This basic patch, which forms an arch spanning the two DBL domains. It is thought that this basic arch interacts with the acidic GPC. It is further believed that this is where RII PfEBA-140 binds GPC because if the molecule is flipped 180°, there is no concentration of positive charges. The basic patch may be better visualized in the picture to the right.<ref name="Lin">PMID: 22989878</ref>
To test whether this basic patch was indeed responsible for erythrocyte binding, two residues in each DBL domain were chosen and mutated to Alanine. In F1, these residues were <scene name='PfEBA-140/Asp247/1'>Asp247</scene> and <scene name='PfEBA-140/Arg254/1'>Arg254</scene>. In F2, these residues were <scene name='PfEBA-140/Arg485/1'>Arg485</scene> and <scene name='PfEBA-140/Asp554/2'>Asp554</scene>. When these four residues were individually mutated to Alanine, erythrocyte binding was heavily interrupted. This can be observed in the figure to the right.<ref name="Lin">PMID: 22989878</ref>[[Image:DecreasedBinding.png | thumb | 100px | alt=text | Mutation of Asp247, Arg254, Arg485, and Asp554]] The decreased erythrocyte binding confirms that these <scene name='PfEBA-140/Four/1'>four residues</scene> found in the basic patch of RII PfEBA-140 are crucial to erythrocyte binding. As a control, residues on the opposite side of RII PfEBA-140 were mutated to Alanine. These residues were Asn394 and Glu605. Mutation of these two residues had no effect on erythrocyte binding.<ref name="Lin">PMID: 22989878</ref>[[Image:FullBinding.png | thumb | 100px | alt=text | Mutation of Asn394 and Glu605]] These results fortify the finding that erythrocyte binding takes place in the basic patch discussed earlier.

 
 
 
 
 

==Structural Differences in PfEBA-140 from other EBL Ligands==
<Structure load='4GF2' size='400' frame='true' align='left' caption='Crystal Structure of PfEBA-140 (PDB entry [[4GF2]])' scene=''/>
There are three unique structural elements that set RII PfEBA-140 apart from other EBL ligands. These structures are the likely reason behind RII PfEBA-140 binding GPC so specifically.
===Disulfide Bonds===
[[Image:ModifiedPattern.png | thumb | 300px | alt=text | Disulfide Bonding Patterns]]
There are twenty-six <scene name='PfEBA-140/Cysteines/1'>cysteines</scene> in RII PfEBA-140, all of which are involved in <scene name='PfEBA-140/Disulfidebonds/1'>disulfide bonding</scene> (disulfide bonds shown in black).<ref name="Lin">PMID: 22989878</ref> In RII PfEBA-140, two modified disulfide patterns were found to be different than in RII PfEBA-175, RII PvDBP, and RII PkDBP. In the latter three RII domains, there is a disulfide linkage between Cys7 and Cys9 and between Cys8 and Cys12. In RII PfEBA-140, however, Cys7 is disulfide bonded to Cys8 and Cys9 is disulfide bonded to Cys12.<ref name="Lin">PMID: 22989878</ref>
===Alpha Helix Structure===
Another difference between RII PfEBA-140 and other EBL ligands can be found in the F2 domain. In EBL ligands of other ''Plasmodium'' species, there are β-fingers with either long or short loops which may or may not be important for receptor binding.<ref name="Lin">PMID: 22989878</ref> While this β-finger is found in F1 of PfEBA-140, it is not found in F2, as it is replaced with an <scene name='PfEBA-140/Alphabeta/1'>α-helix</scene>.<ref name="Lin">PMID: 22989878</ref>
===Lack of Kink in F1===
A third difference between RII PfEBA-140 and other EBL ligands is the lack of a glycine residue in F1. This glycine is present in F1 PfEBA-175, F2 PfEBA-175, PvDBP, PkDBP-α, and F2 PfEBA-140. In F2 PfEBA-140, this glycine is residue 627.<ref name="Lin">PMID: 22989878</ref> In these EBL ligands create a kink due to glycine's flexibility. If this glycine residue were present in F1 of PfEBA-140, it would result in a kink in subdomain 3.<ref name="Lin">PMID: 22989878</ref> This kink would create a hinge angle between the two DBL domains that would allow for dimerization of PfEBA-140 upon erythrocyte binding. For this reason, PfEBA-140 is believed to engage erythrocytes as a monomer, unlike other EBL ligands, which bind erythrocytes as a dimer.
 
 
These three structural differences are believed to be responsible for the specificity of RII PfEBA-140.

==Importance to the Future of Malaria==
RII PfEBA-140 binds irreversibly to erythrocytes, creating a tight junction between the ''Plasmodium falciparum'' merozoites and the host red blood cell. Although more research needs to be performed, PfEBA-140 would serve as a good target for future malaria vaccines as it has been found to be immunogenic.<ref name="Ford">PMID: 17984363</ref>
==References==
<references />

Malaria Parasite Plasmodium falciparum Erythrocyte Binding Antigen 140

2012-11-15T09:09:55Z

Emily Lum:

== Introduction ==
In 2010, malaria caused over 650,000 deaths.<ref name="World Health Organization">http://www.who.int/mediacentre/factsheets/fs094/en/index.html</ref> While this disease is both preventable and curable, many of those that live in areas where the disease is endemic do not have access to such resources. Although there is a vaccine currently undergoing clinical trials, results are not expected until 2014.<ref name="World Health Organization">http://www.who.int/mediacentre/factsheets/fs094/en/index.html</ref> The most recent vaccine research has focused on understanding a group of proteins in the erythrocyte-binding ligand (EBL) family. These proteins, which are found in the ''Plasmodium'' species, facilitate entry into erythrocytes during malarial infection by creating tight junctions between the host erythrocytes and parasite membranes. <ref name="Lin">PMID: 22989878</ref> There are four family members: erythrocyte-binding antigen 175 (PfEBA-175), erythrocyte-binding ligand 1 (PfEBL-1), erythrocyte-binding antigen 140 (PfEBA-140), and erythrocyte-binding antigen 181 (PfEBA-181).<ref name="Lin">PMID: 22989878</ref> PfEBA-140 binds glycophorin C on host erythrocytes,<ref name="Lin">PMID: 22989878</ref> which helps maintain erythrocyte shape and regulates membrane material properties.<ref name="Glycophorin C">http://en.wikipedia.org/wiki/Glycophorin_C</ref> Understanding the mechanism by which PfEBA-140 recognizes and engages glycophorin C on erythrocytes may lead to the future development of a new malaria vaccine.

==General Structure==
<Structure load='4GF2' size='400' frame='true' align='left' caption='Crystal Structure of PfEBA-140 (PDB entry [[4GF2]])' scene=''/>
[[Image:SubdomainsF1F2.png | thumb | 300px | alt=text | Subdomains of F1 and F2]]
[[Image:IndividualSubdomains.png | thumb | 300px | alt=text | Subdomains of F1 and F2]]
The EBL family members, including PfEBA-140, are made up of two regions, region II (RII) (shown to the right) and region VI.<ref name="Lin">PMID: 22989878</ref> Region II is responsible for receptor binding in all EBL family members.<ref name="Lin">PMID: 22989878</ref> RII is composed of <scene name='PfEBA-140/Rii/1'>two Duffy binding-like (DBL) domains</scene>, F1 (purple) and F2 (teal).<ref name="Lin">PMID: 22989878</ref> These two DBL domains are connected by a <scene name='PfEBA-140/Helical_linker/2'>short helical linker</scene>.<ref name="Lin">PMID: 22989878</ref> The DBL protein fold is unique to the ''Plasmodium'' species. Not only does it have the ability to recognize and bind many erythrocyte cell receptors, but it also mediates microvasculature adherence of infected erythrocytes by erythrocyte membrane protein 1 (PfEMP1).<ref name="Lin">PMID: 22989878</ref> Each DBL domain is composed of three subdomains, illustrated in the pictures below.

In the top image, the subdomains S1, S2, and S3 of each of the F1 and F2 domains, as well as the helical linker, are illustrated.<ref name="Lin">PMID: 22989878</ref>
In the bottom image, structures of the individual subdomains are illustrated.<ref name="Lin">PMID: 22989878</ref>

The colors are the same in both images: F1 subdomain 1 is shown in bronze, subdomain 2 in orange, subdomain 3 in dark orange; F2 subdomain 1 is shown in dark blue, subdomain 2 in blue, and subdomain 3 in light blue.

==Binding of RII PfEBA-140==
<Structure load='4GF2' size='400' frame='true' align='left' caption='Crystal Structure of PfEBA-140 (PDB entry [[4GF2]])' scene=''/>
===Requirement of Both DBL Domains===
[[Image:Binding.png | thumb | 100px | alt=text | Erythrocyte Binding of Full Length and Individual RII Domains]]Since the DBL domains of RII are highly conserved, the ability of the individual DBL domains to bind erythrocytes was tested. To do this, constructs containing the full-length RII PfEBA-140 and each individual DBL domain were tested using a rosetting assay. Both brightfield microscopy and green fluorescence protein (GFP) were used to visualize erythrocyte binding. In the upper panel, a construct expressing only GFP was used as a control. The lower panel of Figure A illustrates the extensive erythrocyte binding of the full length RII construct. In Figure B, the lack of black dots in the brightfield microscopy and the smaller quantity of green fluorescence illustrates that F1 and F2 are unable to independently bind erythrocytes. This result suggests that both domains equally participate in engaging erythrocytes.<ref name="Lin">PMID: 22989878</ref>
===Basic Patch===
[[Image:Basic Patch.png | thumb | 100px | alt=text | Erythrocyte Binding of Full Length and Individual RII Domains]]
Since the two DBL domains bind acidic GPC, there must be a number of basic charges that interact with the acidic GPC residues. This idea is supported by visualizing the <scene name='PfEBA-140/Basic_patch/1'>positive and negative residues</scene> found in RII PfEBA-140. The positive side chain residues are red while the negative side chain residues are blue. On the side shown, there are a number of residues with positive side chains, creating a basic patch. This basic patch, which forms an arch spanning the two DBL domains. It is thought that this basic arch interacts with the acidic GPC. It is further believed that this is where RII PfEBA-140 binds GPC because if the molecule is flipped 180°, there is no concentration of positive charges. The basic patch may be better visualized in the picture to the right.<ref name="Lin">PMID: 22989878</ref>
To test whether this basic patch was indeed responsible for erythrocyte binding, two residues in each DBL domain were chosen and mutated to Alanine. In F1, these residues were <scene name='PfEBA-140/Asp247/1'>Asp247</scene> and <scene name='PfEBA-140/Arg254/1'>Arg254</scene>. In F2, these residues were <scene name='PfEBA-140/Arg485/1'>Arg485</scene> and <scene name='PfEBA-140/Asp554/2'>Asp554</scene>. When these four residues were individually mutated to Alanine, erythrocyte binding was heavily interrupted. This can be observed in the figure to the right.<ref name="Lin">PMID: 22989878</ref>[[Image:DecreasedBinding.png | thumb | 100px | alt=text | Mutation of Asp247, Arg254, Arg485, and Asp554]] The decreased erythrocyte binding confirms that these <scene name='PfEBA-140/Four/1'>four residues</scene> found in the basic patch of RII PfEBA-140 are crucial to erythrocyte binding. As a control, residues on the opposite side of RII PfEBA-140 were mutated to Alanine. These residues were Asn394 and Glu605. Mutation of these two residues had no effect on erythrocyte binding.<ref name="Lin">PMID: 22989878</ref>[[Image:FullBinding.png | thumb | 100px | alt=text | Mutation of Asn394 and Glu605]] These results fortify the finding that erythrocyte binding takes place in the basic patch discussed earlier.

 
 
 
 
 

==Structural Differences in PfEBA-140 from other EBL Ligands==
<Structure load='4GF2' size='400' frame='true' align='left' caption='Crystal Structure of PfEBA-140 (PDB entry [[4GF2]])' scene=''/>
There are three unique structural elements that set RII PfEBA-140 apart from other EBL ligands. These structures are the likely reason behind RII PfEBA-140 binding GPC so specifically.
===Disulfide Bonds===
[[Image:ModifiedPattern.png | thumb | 300px | alt=text | Disulfide Bonding Patterns]]
There are twenty-six <scene name='PfEBA-140/Cysteines/1'>cysteines</scene> in RII PfEBA-140, all of which are involved in <scene name='PfEBA-140/Disulfidebonds/1'>disulfide bonding</scene> (disulfide bonds shown in black).<ref name="Lin">PMID: 22989878</ref> In RII PfEBA-140, two modified disulfide patterns were found to be different than in RII PfEBA-175, RII PvDBP, and RII PkDBP. In the latter three RII domains, there is a disulfide linkage between Cys7 and Cys9 and between Cys8 and Cys12. In RII PfEBA-140, however, Cys7 is disulfide bonded to Cys8 and Cys9 is disulfide bonded to Cys12.<ref name="Lin">PMID: 22989878</ref>
===Alpha Helix Structure===
Another difference between RII PfEBA-140 and other EBL ligands can be found in the F2 domain. In EBL ligands of other ''Plasmodium'' species, there are β-fingers with either long or short loops which may or may not be important for receptor binding.<ref name="Lin">PMID: 22989878</ref> While this β-finger is found in F1 of PfEBA-140, it is not found in F2, as it is replaced with an <scene name='PfEBA-140/Alphabeta/1'>α-helix</scene>.<ref name="Lin">PMID: 22989878</ref>
===Lack of Kink in F1===
A third difference between RII PfEBA-140 and other EBL ligands is the lack of a glycine residue in F1. This glycine is present in F1 PfEBA-175, F2 PfEBA-175, PvDBP, PkDBP-α, and F2 PfEBA-140. In F2 PfEBA-140, this glycine is residue 627.<ref name="Lin">PMID: 22989878</ref> In these EBL ligands create a kink due to glycine's flexibility. If this glycine residue were present in F1 of PfEBA-140, it would result in a kink in subdomain 3.<ref name="Lin">PMID: 22989878</ref> This kink would create a hinge angle between the two DBL domains that would allow for dimerization of PfEBA-140 upon erythrocyte binding. For this reason, PfEBA-140 is believed to engage erythrocytes as a monomer, unlike other EBL ligands, which bind erythrocytes as a dimer.
 
 
These three structural differences are believed to be responsible for the specificity of RII PfEBA-140.

==Importance to the Future of Malaria==
RII PfEBA-140 binds irreversibly to erythrocytes, creating a tight junction between the ''Plasmodium falciparum'' merozoites and the host red blood cell. Although more research needs to be performed, PfEBA-140 would serve as a good target for future malaria vaccines as it has been found to be immunogenic.<ref name="Ford">PMID: 17984363</ref>
==References==
<references />

File:SubdomainsF1F2.png

2012-11-15T08:52:03Z

Emily Lum:

== Summary ==
Subdomains S1, S2, and S3 found in F1 and F2.<ref name="Lin">PMID: 22989878</ref>
== Licensing ==
{{subst:Non-commercial from license selector}}

==References==
<references />

File:IndividualSubdomains.png

2012-11-15T08:51:24Z

Emily Lum:

== Summary ==
Subdomains of RII PfEBA-140.<ref name="Lin">PMID: 22989878</ref>
== Licensing ==
{{subst:Non-commercial from license selector}}
==References==
<references />

File:FullBinding.png

2012-11-15T08:50:35Z

Emily Lum:

== Licensing ==
{{subst:Non-commercial from license selector}}<ref name="Lin">PMID: 22989878</ref>
==References==
<references />

File:DecreasedBinding.png

2012-11-15T08:50:00Z

Emily Lum:

== Licensing ==
{{subst:Non-commercial from license selector}}<ref name="Lin">PMID: 22989878</ref>

==References==
<references />

2012-11-15T06:57:15Z

Emily Lum:

File:FullBinding.png

2012-11-15T06:54:35Z

Emily Lum:

== Licensing ==
{{subst:Non-commercial from license selector}}

Malaria Parasite Plasmodium falciparum Erythrocyte Binding Antigen 140

2012-11-15T06:48:21Z

Emily Lum:

File:DecreasedBinding.png

2012-11-15T06:38:58Z

Emily Lum:

== Licensing ==
{{subst:Non-commercial from license selector}}

File:Basic Patch.png

2012-11-15T05:55:59Z

Emily Lum: Basic patch of RII PfEBA-140.

== Summary ==
Basic patch of RII PfEBA-140.
== Licensing ==
{{subst:Non-commercial from license selector}}

Malaria Parasite Plasmodium falciparum Erythrocyte Binding Antigen 140

2012-11-15T05:04:29Z

Emily Lum: