Proteopedia - User contributions [en]

User:Marvin O'Neal/OspB

2012-05-07T16:49:38Z

Olivia Cheng:

Outer Surface Protein B (OspB) has been found to play a vital role in the adherence of B. bugdorferi onto tick guts, which promote the survival of the vector and spread of Lyme disease. OspB-deficient B. budgdorferi have been found to bind poorly to tick gut extracts. The expression of OspB, along with OspA, is upregulated and downregulated by B. Burgdorferi according to the distinct phase of the life cycle that it is in. When the spirochete resides inside the arthropod vector, OspB is upregulated to promote binding to the tick’s gut. However, during transmission from the tick to a vertebrate host, OspB is downregulated and other proteins such as OspC, DpbA and BBK32 are upregulated <ref>PMID: 17352535 </ref>.

= Background =
<Structure load='1rjl' size='450' frame='true' align='right' caption='OspB interacting with Fab H6831' scene='User:Olivia_Cheng/Sandbox_1/1rjl_color/1' />

Lyme disease, discovered in 1975, is spread by means of a tick vector that carries the causative agent, spirochete [http://en.wikipedia.org/wiki/Borrelia_burgdorferi ''Borrelia Burgdorferi''] <ref name="Structural">PMID:15864264</ref>. It is the most frequently reported vectorborne illness in the United States. Individuals who have this disease may show symptoms including skin lesions called erythema migans and the characteristic bulls-eye rash <ref>http://www.cdc.gov/lyme/stats/index.html</ref>. OspA and OspB are the major outer surface proteins found on ''B. Borrelia''. OspB has shown significant variability in amino acid sequence and antigen reactivity in comparison to OspA, known to be largely invariant <ref name="Versatile">PMID:15713683</ref>.

= OspB Interaction with Fab of H6831 =
The [http://en.wikipedia.org/wiki/Lyme_disease_microbiology#Outer_surface_proteins outer-surface proteins] (Osps) in ''B. burgdorferi'' spirochete activate the classical and alternative pathways of the complement system. B. burgdorferi is resistant to complement mediated lysis. The complement inhibitor factor H binds to Osps and the C3b cascade is deactivated <ref name="Versatile"/>.

Within this complex is <scene name='User:Olivia_Cheng/Sandbox_1/1rjl_chainc_transparentabd/6'>a truncated form of OspB</scene>, shown in purple. The H6831 Fab is shown in white. (Click <scene name='User:Olivia_Cheng/Sandbox_1/1rjl_color/1'>here</scene> to revert back to the original.)

H6831 is an IgG class monoclonal complement-independent antibody shown to effectively lyse outer surface protein B (OspB) of ''B. burgdorferi''. H6831 recognizes <scene name='User:Olivia_Cheng/Sandbox_1/1rjl_chainc_lys253/7'>Lys-253</scene> on OspB. Studies have shown that ''B. burgdorferi'' strains with Thr, Cys, Gly, or Glu instead of Lys decrease the binding affinity between H6831 and OspB. The sequence and structure of bactericidal H6831 Fab are typical for IgG2 heavy chain/kappa light chain class. <ref name="Versatile"/>.

The H6831 epitope of OspB is topologically equivalent to LA-2 epitope of OspA <ref name="Versatile"/>. Similar to the LA-2 epitope, the H6831 epitope is positioned opposite the N-terminus near the end of the antigen. The buried surface area of OspB in the H6831 Fab complex is smaller than that of the OspA-LA2 complex. Loop 1 in the OspA-LA2 complex has the most interactions with the Fab, where as Loop 1 in the OspB-H6831 complex has the fewest interactions with the Fab <ref>PMID: 9038292 </ref>.

== Binding ==
<Structure load='1P4P' size='250' frame='true' align='right' caption='Proteolysed Portion of OspB' scene='User:Stephanie_Maung/Sandbox/Gray_1p4p/1' />
H6831 Fab complex binds to a highly accessible region near the C-terminus of OspB, away from the N-terminus lipid anchor. The interaction of OspB-Fab complex depends heavily on hydrogen bonding between <scene name='User:Stephanie_Maung/Sandbox/1p4p_proteolysed_region_loops/5'>loops 1, 2, and 3</scene>. When binding of full-length OspB to Fab fragments of H6831 or CB2 fail, it is usually because <scene name='User:Stephanie_Maung/Sandbox/1p4p_proteolysed_region_loops/3'>Lys-253</scene>, located on loop 2, has been replaced with a different residue. Studies show that substitutions in basic residues of hen egg-white lysozyme (HEL) that participate in HEL-Fab complexes decreased binding affinity by 400-10,000 times. <ref name="Versatile"/>

Due to its effective bactericidal actions, H6831 is used to generate less virulent escape variants of ''B. burgdorferi'' <ref name="Versatile"/>. In the majority of the mutations created from in vivo and in vitro immunization of mice, truncated forms of OspB within the C terminus lead to premature stop codons<ref>PMID:8308101 </ref>. It has been suggested that OspB mutants are more sensitive to proteolysis due to missense mutations that disturb the conformation of OspB <ref name="Versatile"/>. Truncated OspBs cease within the two C-terminal beta-strands of the central sheet. H6831 disorders or removes a beta sheet from OspB after binding. Cleavage may be a possible explanation for the conformational changes of OspB <ref>PMID:9125579</ref>. In <scene name='User:Stephanie_Maung/Sandbox/Gray_1p4p/1'>H-6831 free</scene> and <scene name='User:Stephanie_Maung/Sandbox/1rjl_chainc/1'>H-6831 bound</scene> forms of OspB, some changes result from proteolysis near the N terminus <ref name="Versatile"/>. Residues 157 - 201 on OspB contain the <scene name='User:Stephanie_Maung/Sandbox/1p4p_proteolysed_region_triad/2'>cleaved region</scene>, shown in plum.

Aromatic residues tyrosine and tryptophan are also present in the OspB-H6831 interaction, a feature found in many antigen-antibody complexes. The Lys-253 residue forms a trans conformation between these aromatic residues of H6831. In the complex structure of the antibody binding site, the electron density is well defined and shows increased contact between Lys-253 and the antigen-binding site of the Fab. Most of the electrostatic and hydrogen-bond interactions occur between loop 2 and the Fab heavy chain <ref name="Structural"/>.

= Potential Mechanisms of Lysis =

== Potential Catalytic Triad ==

<Structure load='1p4p' size='250' frame='true' align='left' caption='OspB' scene='Insert optional scene name here' />

[[Image: Cataly.png|300px|right|thumb| Comparison of catalytic triads]]
The mechanism by which H6831 Fab destroys a spirochete appears to be a novel interaction. It is possible that Fab binding changes the properties of OspB folding, which may increase sensitivity of the protein to proteolysis or aggregation. NMR methods showed that the effects of binding can be sent to regions of the antigen distant from epitope, which is at the <scene name='User:Olivia_Cheng/Sandbox_1/1p4p_rainbow/1'>C-terminus</scene> shown in red (N-terminus in blue). OspB shows signs of truncation after interacting with Fab of H6831 <ref>PMID: 1382591</ref>.

It is possible that OspB performs an autoproteolysis. There is a <scene name='User:Olivia_Cheng/Sandbox_1/1p4p_cat_triad_cool/9'>set of three residues</scene> found on OspB that resembles the catalytic triad of [[Serine_Proteases]]. This "constellation" consists of Thr-166, Arg-162, and Glu-184, which is similar to the catalytic triad residues of the serine protease [[trypsin]], which are Ser-195, His-57, Asp-102 <ref> PMID:12475199</ref>.

Threonine and Glutamic acid are found in other catalytic triads of the serine hydrolase family, but argenine seems unlikely to replace histidine as a base because of its higher pKa. There have been studies that have shown that Argenine is essential for other enzymatic functions, such as in the Ser-Arg-Asp triad in cytosolic phospholipase A2 and as a catalytic base in Sortase A. <scene name='User:Olivia_Cheng/Sandbox_1/1p4p_asn164/1'>Asn-164</scene> forms an H-bond with <scene name='User:Olivia_Cheng/Sandbox_1/1p4p_asn164/2'>Thr-166</scene> and may rearrange to form a putative oxyanion hole with Thr-166 and another unidentified atom if active in the catalysis. A concerted proton transfer, similar to a “proton wire”, is one plausible mechanism that would allow argenine to function in the catalytic triad of a protease <ref name="Structural"/>.

== Potential Oxidative Mechanism ==
It was recently discovered that all antibodies contained Fab portions that catalyzed a reaction between singlet oxygen and water, yielding hydrogen peroxide, ozone, water and hydroxide radicals. Hydrogen peroxide is a toxic oxidative species and might be the product of an ancient mechanism to protect against infection. UV absorption increases the rate for this reaction. B. burgdorferi is especially vulnerable to oxidative damage because its ecological niche is in areas with limited oxygen and its genome does not encode a catalase. This oxidative mechanism might explain why some mABs are bactericidal without the use of complement <ref name="Structural"/>.

= 3D Structures =

[[Monoclonal_Antibody]]
 
[[Trypsin]]

= Links =

*[http://www.cdc.gov/lyme/ cdc.gov/lyme/] - the Official Lyme Disease resource of the US Center for Disease Control.

= References =

<references />

User:Olivia Cheng/Sandbox 1

2012-05-04T21:00:30Z

Olivia Cheng:

User:Olivia Cheng/Sandbox 1

2012-05-04T20:58:55Z

Olivia Cheng:

Outer Surface Protein B (OspB) has been found to play a vital role in the adherence of B. bugdorferi onto tick guts, which promote the survival of the vector and spread of Lyme disease. OspB-deficient B. budgdorferi have been found to bind poorly to tick gut extracts. The expression of OspB, along with OspA, is upregulated and downregulated by B. Burgdorferi according to the distinct phase of the life cycle that it is in. When the spirochete resides inside the arthropod vector, OspB is upregulated to promote binding to the tick’s gut. However, during transmission from the tick to a vertebrate host, OspB is downregulated and other proteins such as OspC, DpbA and BBK32 are upregulated <ref>PMID: 17352535 </ref>.

= Background =
<Structure load='1rjl' size='450' frame='true' align='right' caption='OspB interacting with Fab H6831' scene='User:Olivia_Cheng/Sandbox_1/1rjl_color/1' />

Lyme disease, discovered in 1975, is spread by means of a tick vector that carries the causative agent, spirochete [http://en.wikipedia.org/wiki/Borrelia_burgdorferi ''Borrelia Burgdorferi''] <ref name="Structural">PMID:15864264</ref>. It is the most frequently reported vectorborne illness in the United States. Individuals who have this disease may show symptoms including skin lesions called erythema migans and the characteristic bulls-eye rash <ref>http://www.cdc.gov/lyme/stats/index.html</ref>. OspA and OspB are the major outer surface proteins found on ''B. Borrelia''. OspB has shown significant variability in amino acid sequence and antigen reactivity in comparison to OspA, known to be largely invariant <ref name="Versatile">PMID:15713683</ref>.

= OspB Interaction with Fab of H6831 =
The [http://en.wikipedia.org/wiki/Lyme_disease_microbiology#Outer_surface_proteins outer-surface proteins] (Osps) in ''B. burgdorferi'' spirochete activate the classical and alternative pathways of the complement system. B. burgdorferi is resistant to complement mediated lysis. The complement inhibitor factor H binds to Osps and the C3b cascade is deactivated <ref name="Versatile"/>.

Within this complex is <scene name='User:Olivia_Cheng/Sandbox_1/1rjl_chainc_transparentabd/6'>a truncated form of OspB</scene>, shown in purple. The H6831 Fab is shown in white. (Click <scene name='User:Olivia_Cheng/Sandbox_1/1rjl_color/1'>here</scene> to revert back to the original.)

H6831 is an IgG class monoclonal complement-independent antibody shown to effectively lyse outer surface protein B (OspB) of ''B. burgdorferi''. H6831 recognizes <scene name='User:Olivia_Cheng/Sandbox_1/1rjl_chainc_lys253/7'>Lys-253</scene> on OspB. Studies have shown that ''B. burgdorferi'' strains with Thr, Cys, Gly, or Glu instead of Lys decrease the binding affinity between H6831 and OspB. The sequence and structure of bactericidal H6831 Fab are typical for IgG2 heavy chain/kappa light chain class. <ref name="Versatile"/>.

The H6831 epitope of OspB is topologically equivalent to LA-2 epitope of OspA <ref name="Versatile"/>. Similar to the LA-2 epitope, the H6831 epitope is positioned opposite the N-terminus near the end of the antigen. The buried surface area of OspB in the H6831 Fab complex is smaller than that of the OspA-LA2 complex. Loop 1 in the OspA-LA2 complex has the most interactions with the Fab, where as Loop 1 in the OspB-H6831 complex has the fewest interactions with the Fab <ref>PMID: 9038292 </ref>.

== Binding ==
<Structure load='1P4P' size='250' frame='true' align='right' caption='Proteolysed Portion of OspB' scene='User:Stephanie_Maung/Sandbox/Gray_1p4p/1' />
H6831 Fab complex binds to a highly accessible region near the C-terminus of OspB, away from the N-terminus lipid anchor. The interaction of OspB-Fab complex depends heavily on hydrogen bonding between <scene name='User:Stephanie_Maung/Sandbox/1p4p_proteolysed_region_loops/5'>loops 1, 2, and 3</scene>. When binding of full-length OspB to Fab fragments of H6831 or CB2 fail, it is usually because <scene name='User:Stephanie_Maung/Sandbox/1p4p_proteolysed_region_loops/3'>Lys-253</scene>, located on loop 2, has been replaced with a different residue. Studies show that substitutions in basic residues of hen egg-white lysozyme (HEL) that participate in HEL-Fab complexes decreased binding affinity by 400-10,000 times. <ref name="Versatile"/>

Due to its effective bactericidal actions, H6831 is used to generate less virulent escape variants of ''B. burgdorferi'' <ref name="Versatile"/>. In the majority of the mutations created from in vivo and in vitro immunization of mice, truncated forms of OspB within the C terminus lead to premature stop codons<ref>PMID:8308101 </ref>. It has been suggested that OspB mutants are more sensitive to proteolysis due to missense mutations that disturb the conformation of OspB <ref name="Versatile"/>. Truncated OspBs cease within the two C-terminal beta-strands of the central sheet. H6831 disorders or removes a beta sheet from OspB after binding. Cleavage may be a possible explanation for the conformational changes of OspB <ref>PMID:9125579</ref>. In <scene name='User:Stephanie_Maung/Sandbox/Gray_1p4p/1'>H-6831 free</scene> and <scene name='User:Stephanie_Maung/Sandbox/1rjl_chainc/1'>H-6831 bound</scene> forms of OspB, some changes result from proteolysis near the N terminus <ref name="Versatile"/>. Residues 157 - 201 on OspB contain the <scene name='User:Stephanie_Maung/Sandbox/1p4p_proteolysed_region_triad/2'>cleaved region</scene>, shown in plum.

Aromatic residues tyrosine and tryptophan are also present in the OspB-H6831 interaction, a feature found in many antigen-antibody complexes. The Lys-253 residue forms a trans conformation between these aromatic residues of H6831. In the complex structure of the antibody binding site, the electron density is well defined and shows increased contact between Lys-253 and the antigen-binding site of the Fab. Most of the electrostatic and hydrogen-bond interactions occur between loop 2 and the Fab heavy chain <ref name="Structural"/>.

= Potential Mechanisms of Lysis =

== Potential Catalytic Triad ==

<Structure load='1p4p' size='250' frame='true' align='left' caption='OspB' scene='Insert optional scene name here' />

[[Image: Cataly.png|300px|right|thumb| Comparison of catalytic triads]]
The mechanism by which H6831 Fab destroys a spirochete appears to be a novel interaction. It is possible that Fab binding changes the properties of OspB folding, which may increase sensitivity of the protein to proteolysis or aggregation. NMR methods showed that the effects of binding can be sent to regions of the antigen distant from epitope, which is at the <scene name='User:Olivia_Cheng/Sandbox_1/1p4p_rainbow/1'>C-terminus</scene> shown in red (N-terminus in blue). OspB shows signs of truncation after interacting with Fab of H6831 <ref>PMID: 1382591</ref>.

It is possible that OspB performs an autoproteolysis. There is a <scene name='User:Olivia_Cheng/Sandbox_1/1p4p_cat_triad_cool/9'>set of three residues</scene> found on OspB that resembles the catalytic triad of [[Serine_Proteases]]. This "constellation" consists of Thr-166, Arg-162, and Glu-184, which is similar to the catalytic triad residues of the serine protease [[trypsin]], which are Ser-195, His-57, Asp-102 <ref> PMID:12475199</ref>.

Threonine and Glutamic acid are found in other catalytic triads of the serine hydrolase family, but argenine seems unlikely to replace histidine as a base because of its higher pKa. There have been studies that have shown that Argenine is essential for other enzymatic functions, such as in the Ser-Arg-Asp triad in cytosolic phospholipase A2 and as a catalytic base in Sortase A. <scene name='User:Olivia_Cheng/Sandbox_1/1p4p_asn164/1'>Asn-164</scene> forms an H-bond with <scene name='User:Olivia_Cheng/Sandbox_1/1p4p_asn164/2'>Thr-166</scene> and may rearrange to form a putative oxyanion hole with Thr-166 and another unidentified atom if active in the catalysis. A concerted proton transfer, similar to a “proton wire”, is one plausible mechanism that would allow argenine to function in the catalytic triad of a protease <ref name="Structural"/>.

== Potential Oxidative Mechanism ==
It was recently discovered that all antibodies contained Fab portions that catalyzed a reaction between singlet oxygen and water, yielding hydrogen peroxide, ozone, water and hydroxide radicals. Hydrogen peroxide is a toxic oxidative species and might be the product of an ancient mechanism to protect against infection. UV absorption increases the rate for this reaction. B. burgdorferi is especially vulnerable to oxidative damage because its ecological niche is in areas with limited oxygen and its genome does not encode a catalase. This oxidative mechanism might explain why some mABs are bactericidal without the use of complement <ref name="Structural"/>.

= 3D Structures =

[[Monoclonal_Antibody]]
 
[[Trypsin]]

= Links =

*[cdc.gov/lyme] - the Official Lyme Disease resource of the US Center for Disease Control.

= References =

<references />

User:Olivia Cheng/Sandbox 1

2012-05-04T20:57:17Z

Olivia Cheng:

Outer Surface Protein B (OspB) has been found to play a vital role in the adherence of B. bugdorferi onto tick guts, which promote the survival of the vector and spread of Lyme disease. OspB-deficient B. budgdorferi have been found to bind poorly to tick gut extracts. The expression of OspB, along with OspA, is upregulated and downregulated by B. Burgdorferi according to the distinct phase of the life cycle that it is in. When the spirochete resides inside the arthropod vector, OspB is upregulated to promote binding to the tick’s gut. However, during transmission from the tick to a vertebrate host, OspB is downregulated and other proteins such as OspC, DpbA and BBK32 are upregulated <ref>PMID: 17352535 </ref>.

= Background =
<Structure load='1rjl' size='450' frame='true' align='right' caption='OspB interacting with Fab H6831' scene='User:Olivia_Cheng/Sandbox_1/1rjl_color/1' />

Lyme disease, discovered in 1975, is spread by means of a tick vector that carries the causative agent, spirochete [http://en.wikipedia.org/wiki/Borrelia_burgdorferi ''Borrelia Burgdorferi''] <ref name="Structural">PMID:15864264</ref>. It is the most frequently reported vectorborne illness in the United States. Individuals who have this disease may show symptoms including skin lesions called erythema migans and the characteristic bulls-eye rash <ref>http://www.cdc.gov/lyme/stats/index.html</ref>. OspA and OspB are the major outer surface proteins found on ''B. Borrelia''. OspB has shown significant variability in amino acid sequence and antigen reactivity in comparison to OspA, known to be largely invariant <ref name="Versatile">PMID:15713683</ref>.

= OspB Interaction with Fab of H6831 =
The [http://en.wikipedia.org/wiki/Lyme_disease_microbiology#Outer_surface_proteins outer-surface proteins] (Osps) in ''B. burgdorferi'' spirochete activate the classical and alternative pathways of the complement system. B. burgdorferi is resistant to complement mediated lysis. The complement inhibitor factor H binds to Osps and the C3b cascade is deactivated <ref name="Versatile"/>.

Within this complex is <scene name='User:Olivia_Cheng/Sandbox_1/1rjl_chainc_transparentabd/6'>a truncated form of OspB</scene>, shown in purple. The H6831 Fab is shown in white. (Click <scene name='User:Olivia_Cheng/Sandbox_1/1rjl_color/1'>here</scene> to revert back to the original.)

H6831 is an IgG class monoclonal complement-independent antibody shown to effectively lyse outer surface protein B (OspB) of ''B. burgdorferi''. H6831 recognizes <scene name='User:Olivia_Cheng/Sandbox_1/1rjl_chainc_lys253/7'>Lys-253</scene> on OspB. Studies have shown that ''B. burgdorferi'' strains with Thr, Cys, Gly, or Glu instead of Lys decrease the binding affinity between H6831 and OspB. The sequence and structure of bactericidal H6831 Fab are typical for IgG2 heavy chain/kappa light chain class. <ref name="Versatile"/>.

The H6831 epitope of OspB is topologically equivalent to LA-2 epitope of OspA <ref name="Versatile"/>. Similar to the LA-2 epitope, the H6831 epitope is positioned opposite the N-terminus near the end of the antigen. The buried surface area of OspB in the H6831 Fab complex is smaller than that of the OspA-LA2 complex. Loop 1 in the OspA-LA2 complex has the most interactions with the Fab, where as Loop 1 in the OspB-H6831 complex has the fewest interactions with the Fab <ref>PMID: 9038292 </ref>.

== Binding ==
<Structure load='1P4P' size='250' frame='true' align='right' caption='Proteolysed Portion of OspB' scene='User:Stephanie_Maung/Sandbox/Gray_1p4p/1' />
H6831 Fab complex binds to a highly accessible region near the C-terminus of OspB, away from the N-terminus lipid anchor. The interaction of OspB-Fab complex depends heavily on hydrogen bonding between <scene name='User:Stephanie_Maung/Sandbox/1p4p_proteolysed_region_loops/5'>loops 1, 2, and 3</scene>. When binding of full-length OspB to Fab fragments of H6831 or CB2 fail, it is usually because <scene name='User:Stephanie_Maung/Sandbox/1p4p_proteolysed_region_loops/3'>Lys-253</scene>, located on loop 2, has been replaced with a different residue. Studies show that substitutions in basic residues of hen egg-white lysozyme (HEL) that participate in HEL-Fab complexes decreased binding affinity by 400-10,000 times. <ref name="Versatile"/>

Due to its effective bactericidal actions, H6831 is used to generate less virulent escape variants of ''B. burgdorferi'' <ref name="Versatile"/>. In the majority of the mutations created from in vivo and in vitro immunization of mice, truncated forms of OspB within the C terminus lead to premature stop codons<ref>PMID:8308101 </ref>. It has been suggested that OspB mutants are more sensitive to proteolysis due to missense mutations that disturb the conformation of OspB <ref name="Versatile"/>. Truncated OspBs cease within the two C-terminal beta-strands of the central sheet. H6831 disorders or removes a beta sheet from OspB after binding. Cleavage may be a possible explanation for the conformational changes of OspB <ref>PMID:9125579</ref>. In <scene name='User:Stephanie_Maung/Sandbox/Gray_1p4p/1'>H-6831 free</scene> and <scene name='User:Stephanie_Maung/Sandbox/1rjl_chainc/1'>H-6831 bound</scene> forms of OspB, some changes result from proteolysis near the N terminus <ref name="Versatile"/>. Residues 157 - 201 on OspB contain the <scene name='User:Stephanie_Maung/Sandbox/1p4p_proteolysed_region_triad/2'>cleaved region</scene>, shown in plum.

Aromatic residues tyrosine and tryptophan are also present in the OspB-H6831 interaction, a feature found in many antigen-antibody complexes. The Lys-253 residue forms a trans conformation between these aromatic residues of H6831. In the complex structure of the antibody binding site, the electron density is well defined and shows increased contact between Lys-253 and the antigen-binding site of the Fab. Most of the electrostatic and hydrogen-bond interactions occur between loop 2 and the Fab heavy chain <ref name="Structural"/>.

= Potential Mechanisms of Lysis =

== Potential Catalytic Triad ==

<Structure load='1p4p' size='250' frame='true' align='left' caption='OspB' scene='Insert optional scene name here' />

[[Image: Cataly.png|300px|right|thumb| Comparison of catalytic triads]]
The mechanism by which H6831 Fab destroys a spirochete appears to be a novel interaction. It is possible that Fab binding changes the properties of OspB folding, which may increase sensitivity of the protein to proteolysis or aggregation. NMR methods showed that the effects of binding can be sent to regions of the antigen distant from epitope, which is at the <scene name='User:Olivia_Cheng/Sandbox_1/1p4p_rainbow/1'>C-terminus</scene> shown in red (N-terminus in blue). OspB shows signs of truncation after interacting with Fab of H6831 <ref>PMID: 1382591</ref>.

It is possible that OspB performs an autoproteolysis. There is a <scene name='User:Olivia_Cheng/Sandbox_1/1p4p_cat_triad_cool/9'>set of three residues</scene> found on OspB that resembles the catalytic triad of [[Serine_Proteases]]. This "constellation" consists of Thr-166, Arg-162, and Glu-184, which is similar to the catalytic triad residues of the serine protease [[trypsin]], which are Ser-195, His-57, Asp-102 <ref> PMID:12475199</ref>.

Threonine and Glutamic acid are found in other catalytic triads of the serine hydrolase family, but argenine seems unlikely to replace histidine as a base because of its higher pKa. There have been studies that have shown that Argenine is essential for other enzymatic functions, such as in the Ser-Arg-Asp triad in cytosolic phospholipase A2 and as a catalytic base in Sortase A. <scene name='User:Olivia_Cheng/Sandbox_1/1p4p_asn164/1'>Asn-164</scene> forms an H-bond with <scene name='User:Olivia_Cheng/Sandbox_1/1p4p_asn164/2'>Thr-166</scene> and may rearrange to form a putative oxyanion hole with Thr-166 and another unidentified atom if active in the catalysis. A concerted proton transfer, similar to a “proton wire”, is one plausible mechanism that would allow argenine to function in the catalytic triad of a protease <ref name="Structural"/>.

== Potential Oxidative Mechanism ==
It was recently discovered that all antibodies contained Fab portions that catalyzed a reaction between singlet oxygen and water, yielding hydrogen peroxide, ozone, water and hydroxide radicals. Hydrogen peroxide is a toxic oxidative species and might be the product of an ancient mechanism to protect against infection. UV absorption increases the rate for this reaction. B. burgdorferi is especially vulnerable to oxidative damage because its ecological niche is in areas with limited oxygen and its genome does not encode a catalase. This oxidative mechanism might explain why some mABs are bactericidal without the use of complement <ref name="Structural"/>.

= 3D Structures =

[[Monoclonal_Antibody]]
 
[[Trypsin]]

= Links =

*[cdc.gov/lyme] - the Official Lyme Disease resource of the US Center for Disease Control.

= References =

<references />

User:Olivia Cheng/Sandbox 1

2012-05-04T20:53:39Z

Olivia Cheng:

Outer Surface Protein B (OspB) has been found to play a vital role in the adherence of B. bugdorferi onto tick guts, which promote the survival of the vector and spread of Lyme disease. OspB-deficient B. budgdorferi have been found to bind poorly to tick gut extracts. The expression of OspB, along with OspA, is upregulated and downregulated by B. Burgdorferi according to the distinct phase of the life cycle that it is in. When the spirochete resides inside the arthropod vector, OspB is upregulated to promote binding to the tick’s gut. However, during transmission from the tick to a vertebrate host, OspB is downregulated and other proteins such as OspC, DpbA and BBK32 are upregulated <ref>PMID: 17352535 </ref>.

= Background =
<Structure load='1rjl' size='450' frame='true' align='right' caption='OspB interacting with Fab H6831' scene='User:Olivia_Cheng/Sandbox_1/1rjl_color/1' />

Lyme disease, discovered in 1975, is spread by means of a tick vector that carries the causative agent, spirochete [http://en.wikipedia.org/wiki/Borrelia_burgdorferi ''Borrelia Burgdorferi''] <ref name="Structural">PMID:15864264</ref>. It is the most frequently reported vectorborne illness in the United States. Individuals who have this disease may show symptoms including skin lesions called erythema migans and the characteristic bulls-eye rash <ref>http://www.cdc.gov/lyme/stats/index.html</ref>. OspA and OspB are the major outer surface proteins found on ''B. Borrelia''. OspB has shown significant variability in amino acid sequence and antigen reactivity in comparison to OspA, known to be largely invariant <ref name="Versatile">PMID:15713683</ref>.

= OspB Interaction with Fab of H6831 =
The [http://en.wikipedia.org/wiki/Lyme_disease_microbiology#Outer_surface_proteins outer-surface proteins] (Osps) in ''B. burgdorferi'' spirochete activate the classical and alternative pathways of the complement system. B. burgdorferi is resistant to complement mediated lysis. The complement inhibitor factor H binds to Osps and the C3b cascade is deactivated <ref name="Versatile"/>.

Within this complex is <scene name='User:Olivia_Cheng/Sandbox_1/1rjl_chainc_transparentabd/6'>a truncated form of OspB</scene>, shown in purple. The H6831 Fab is shown in white. (Click <scene name='User:Olivia_Cheng/Sandbox_1/1rjl_color/1'>here</scene> to revert back to the original.)

H6831 is an IgG class monoclonal complement-independent antibody shown to effectively lyse outer surface protein B (OspB) of ''B. burgdorferi''. H6831 recognizes <scene name='User:Olivia_Cheng/Sandbox_1/1rjl_chainc_lys253/6'>Lys-253</scene> on OspB. Studies have shown that ''B. burgdorferi'' strains with Thr, Cys, Gly, or Glu instead of Lys decrease the binding affinity between H6831 and OspB. The sequence and structure of bactericidal H6831 Fab are typical for IgG2 heavy chain/kappa light chain class. <ref name="Versatile"/>.

The H6831 epitope of OspB is topologically equivalent to LA-2 epitope of OspA <ref name="Versatile"/>. Similar to the LA-2 epitope, the H6831 epitope is positioned opposite the N-terminus near the end of the antigen. The buried surface area of OspB in the H6831 Fab complex is smaller than that of the OspA-LA2 complex. Loop 1 in the OspA-LA2 complex has the most interactions with the Fab, where as Loop 1 in the OspB-H6831 complex has the fewest interactions with the Fab <ref>PMID: 9038292 </ref>.

== Binding ==
<Structure load='1P4P' size='250' frame='true' align='right' caption='Proteolysed Portion of OspB' scene='User:Stephanie_Maung/Sandbox/Gray_1p4p/1' />

H6831 Fab complex binds to a highly accessible region near the C-terminus of OspB, away from the N-terminus lipid anchor. The interaction of OspB-Fab complex depends heavily on hydrogen bonding between <scene name='User:Stephanie_Maung/Sandbox/1p4p_proteolysed_region_loops/5'>loops 1, 2, and 3</scene>. When binding of full-length OspB to Fab fragments of H6831 or CB2 fail, it is usually because <scene name='User:Stephanie_Maung/Sandbox/1p4p_proteolysed_region_loops/3'>Lys-253</scene>, located on loop 2, has been replaced with a different residue. Studies show that substitutions in basic residues of hen egg-white lysozyme (HEL) that participate in HEL-Fab complexes decreased binding affinity by 400-10,000 times. <ref name="Versatile"/>

Due to its effective bactericidal actions, H6831 is used to generate less virulent escape variants of ''B. burgdorferi'' <ref name="Versatile"/>. In the majority of the mutations created from in vivo and in vitro immunization of mice, truncated forms of OspB within the C terminus lead to premature stop codons<ref>PMID:8308101 </ref>. It has been suggested that OspB mutants are more sensitive to proteolysis due to missense mutations that disturb the conformation of OspB <ref name="Versatile"/>. Truncated OspBs cease within the two C-terminal beta-strands of the central sheet. H6831 disorders or removes a beta sheet from OspB after binding. Cleavage may be a possible explanation for the conformational changes of OspB <ref>PMID:9125579</ref>. In <scene name='User:Stephanie_Maung/Sandbox/Gray_1p4p/1'>H-6831 free</scene> and <scene name='User:Stephanie_Maung/Sandbox/1rjl_chainc/1'>H-6831 bound</scene> forms of OspB, some changes result from proteolysis near the N terminus <ref name="Versatile"/>. Residues 157 - 201 on OspB contain the <scene name='User:Stephanie_Maung/Sandbox/1p4p_proteolysed_region_triad/2'>cleaved region</scene>, shown in plum.

Aromatic residues tyrosine and tryptophan are also present in the OspB-H6831 interaction, a feature found in many antigen-antibody complexes. The Lys-253 residue forms a trans conformation between these aromatic residues of H6831. In the complex structure of the antibody binding site, the electron density is well defined and shows increased contact between Lys-253 and the antigen-binding site of the Fab. Most of the electrostatic and hydrogen-bond interactions occur between loop 2 and the Fab heavy chain <ref name="Structural"/>.

= Potential Mechanisms of Lysis =

== Potential Catalytic Triad ==

<Structure load='1p4p' size='250' frame='true' align='left' caption='OspB' scene='Insert optional scene name here' />

[[Image: Cataly.png|300px|right|thumb| Comparison of catalytic triads]]

The mechanism by which H6831 Fab destroys a spirochete appears to be a novel interaction. It is possible that Fab binding changes the properties of OspB folding, which may increase sensitivity of the protein to proteolysis or aggregation. NMR methods showed that the effects of binding can be sent to regions of the antigen distant from epitope, which is at the <scene name='User:Olivia_Cheng/Sandbox_1/1p4p_rainbow/1'>C-terminus</scene> shown in red (N-terminus in blue). OspB shows signs of truncation after interacting with Fab of H6831 <ref>PMID: 1382591</ref>.

It is possible that OspB performs an autoproteolysis. There is a <scene name='User:Olivia_Cheng/Sandbox_1/1p4p_cat_triad_cool/9'>set of three residues</scene> found on OspB that resembles the catalytic triad of [[Serine_Proteases]]. This "constellation" consists of Thr-166, Arg-162, and Glu-184, which is similar to the catalytic triad residues of the serine protease [[trypsin]], which are Ser-195, His-57, Asp-102 <ref> PMID:12475199</ref>.

Threonine and Glutamic acid are found in other catalytic triads of the serine hydrolase family, but argenine seems unlikely to replace histidine as a base because of its higher pKa. There have been studies that have shown that Argenine is essential for other enzymatic functions, such as in the Ser-Arg-Asp triad in cytosolic phospholipase A2 and as a catalytic base in Sortase A. <scene name='User:Olivia_Cheng/Sandbox_1/1p4p_asn164/1'>Asn-164</scene> forms an H-bond with <scene name='User:Olivia_Cheng/Sandbox_1/1p4p_asn164/2'>Thr-166</scene> and may rearrange to form a putative oxyanion hole with Thr-166 and another unidentified atom if active in the catalysis. A concerted proton transfer, similar to a “proton wire”, is one plausible mechanism that would allow argenine to function in the catalytic triad of a protease <ref name="Structural"/>.

== Potential Oxidative Mechanism ==
It was recently discovered that all antibodies contained Fab portions that catalyzed a reaction between singlet oxygen and water, yielding hydrogen peroxide, ozone, water and hydroxide radicals. Hydrogen peroxide is a toxic oxidative species and might be the product of an ancient mechanism to protect against infection. UV absorption increases the rate for this reaction. B. burgdorferi is especially vulnerable to oxidative damage because its ecological niche is in areas with limited oxygen and its genome does not encode a catalase. This oxidative mechanism might explain why some mABs are bactericidal without the use of complement <ref name="Structural"/>.

= 3D Structures =

[[Monoclonal_Antibody]]
 
[[Trypsin]]

= Links =

*[cdc.gov/lyme] - the Official Lyme Disease resource of the US Center for Disease Control.

= References =

<references />

User:Olivia Cheng/Sandbox 1

2012-05-04T20:51:22Z

Olivia Cheng:

Outer Surface Protein B (OspB) has been found to play a vital role in the adherence of B. bugdorferi onto tick guts, which promote the survival of the vector and spread of Lyme disease. OspB-deficient B. budgdorferi have been found to bind poorly to tick gut extracts. The expression of OspB, along with OspA, is upregulated and downregulated by B. Burgdorferi according to the distinct phase of the life cycle that it is in. When the spirochete resides inside the arthropod vector, OspB is upregulated to promote binding to the tick’s gut. However, during transmission from the tick to a vertebrate host, OspB is downregulated and other proteins such as OspC, DpbA and BBK32 are upregulated <ref>PMID: 17352535 </ref>.

= Background =
<Structure load='1rjl' size='450' frame='true' align='right' caption='OspB interacting with Fab H6831' scene='User:Olivia_Cheng/Sandbox_1/1rjl_color/1' />

Lyme disease, discovered in 1975, is spread by means of a tick vector that carries the causative agent, spirochete [http://en.wikipedia.org/wiki/Borrelia_burgdorferi ''Borrelia Burgdorferi''] <ref name="Structural">PMID:15864264</ref>. It is the most frequently reported vectorborne illness in the United States. Individuals who have this disease may show symptoms including skin lesions called erythema migans and the characteristic bulls-eye rash <ref>http://www.cdc.gov/lyme/stats/index.html</ref>. OspA and OspB are the major outer surface proteins found on ''B. Borrelia''. OspB has shown significant variability in amino acid sequence and antigen reactivity in comparison to OspA, known to be largely invariant <ref name="Versatile">PMID:15713683</ref>.

= OspB Interaction with Fab of H6831 =
The [http://en.wikipedia.org/wiki/Lyme_disease_microbiology#Outer_surface_proteins outer-surface proteins] (Osps) in ''B. burgdorferi'' spirochete activate the classical and alternative pathways of the complement system. B. burgdorferi is resistant to complement mediated lysis. The complement inhibitor factor H binds to Osps and the C3b cascade is deactivated <ref name="Versatile"/>.

Within this complex is <scene name='User:Olivia_Cheng/Sandbox_1/1rjl_chainc_transparentabd/6'>a truncated form of OspB</scene>, shown in purple. The H6831 Fab is shown in white. (Click <scene name='User:Olivia_Cheng/Sandbox_1/1rjl_color/1'>here</scene> to revert back to the original.)

H6831 is an IgG class monoclonal complement-independent antibody shown to effectively lyse outer surface protein B (OspB) of ''B. burgdorferi''. H6831 recognizes <scene name='User:Olivia_Cheng/Sandbox_1/1rjl_chainc_lys253/6'>Lys-253</scene> on OspB. Studies have shown that ''B. burgdorferi'' strains with Thr, Cys, Gly, or Glu instead of Lys decrease the binding affinity between H6831 and OspB. The sequence and structure of bactericidal H6831 Fab are typical for IgG2 heavy chain/kappa light chain class. <ref name="Versatile"/>.

The H6831 epitope of OspB is topologically equivalent to LA-2 epitope of OspA <ref name="Versatile"/>. Similar to the LA-2 epitope, the H6831 epitope is positioned opposite the N-terminus near the end of the antigen. The buried surface area of OspB in the H6831 Fab complex is smaller than that of the OspA-LA2 complex. Loop 1 in the OspA-LA2 complex has the most interactions with the Fab, where as Loop 1 in the OspB-H6831 complex has the fewest interactions with the Fab <ref>PMID: 9038292 </ref>.

== Binding ==
<Structure load='1P4P' size='250' frame='true' align='right' caption='Proteolysed Portion of OspB' scene='User:Stephanie_Maung/Sandbox/Gray_1p4p/1' />

H6831 Fab complex binds to a highly accessible region near the C-terminus of OspB, away from the N-terminus lipid anchor. The interaction of OspB-Fab complex depends heavily on hydrogen bonding between <scene name='User:Stephanie_Maung/Sandbox/1p4p_proteolysed_region_loops/5'>loops 1, 2, and 3</scene>. When binding of full-length OspB to Fab fragments of H6831 or CB2 fail, it is usually because <scene name='User:Stephanie_Maung/Sandbox/1p4p_proteolysed_region_loops/3'>Lys-253</scene>, located on loop 2, has been replaced with a different residue. Studies show that substitutions in basic residues of hen egg-white lysozyme (HEL) that participate in HEL-Fab complexes decreased binding affinity by 400-10,000 times. <ref name="Versatile"/>

Due to its effective bactericidal actions, H6831 is used to generate less virulent escape variants of ''B. burgdorferi'' <ref name="Versatile"/>. In the majority of the mutations created from in vivo and in vitro immunization of mice, truncated forms of OspB within the C terminus lead to premature stop codons<ref>PMID:8308101 </ref>. It has been suggested that OspB mutants are more sensitive to proteolysis due to missense mutations that disturb the conformation of OspB <ref name="Versatile"/>. Truncated OspBs cease within the two C-terminal beta-strands of the central sheet. H6831 disorders or removes a beta sheet from OspB after binding. Cleavage may be a possible explanation for the conformational changes of OspB <ref>PMID:9125579</ref>. In <scene name='User:Stephanie_Maung/Sandbox/Gray_1p4p/1'>H-6831 free</scene> and <scene name='User:Stephanie_Maung/Sandbox/1rjl_chainc/1'>H-6831 bound</scene> forms of OspB, some changes result from proteolysis near the N terminus <ref name="Versatile"/>. Residues 157 - 201 on OspB contain the <scene name='User:Stephanie_Maung/Sandbox/1p4p_proteolysed_region_triad/2'>cleaved region</scene>, shown in plum.

Aromatic residues tyrosine and tryptophan are also present in the OspB-H6831 interaction, a feature found in many antigen-antibody complexes. The Lys-253 residue forms a trans conformation between these aromatic residues of H6831. In the complex structure of the antibody binding site, the electron density is well defined and shows increased contact between Lys-253 and the antigen-binding site of the Fab. Most of the electrostatic and hydrogen-bond interactions occur between loop 2 and the Fab heavy chain <ref name="Structural"/>.

= Potential Mechanisms of Lysis =

== Potential Catalytic Triad ==

<Structure load='1p4p' size='250' frame='true' align='left' caption='OspB' scene='Insert optional scene name here' />

[[Image: Cataly.png|300px|right|thumb| Comparison of catalytic triads]]

The mechanism by which H6831 Fab destroys a spirochete appears to be a novel interaction. It is possible that Fab binding changes the properties of OspB folding, which may increase sensitivity of the protein to proteolysis or aggregation. NMR methods showed that the effects of binding can be sent to regions of the antigen distant from epitope, which is at the <scene name='User:Olivia_Cheng/Sandbox_1/1p4p_rainbow/1'>C-terminus</scene> shown in blue (N-terminus in red). OspB shows signs of truncation after interacting with Fab of H6831 <ref>PMID: 1382591</ref>.

It is possible that OspB performs an autoproteolysis. There is a <scene name='User:Olivia_Cheng/Sandbox_1/1p4p_cat_triad_cool/9'>set of three residues</scene> found on OspB that resembles the catalytic triad of [[Serine_Proteases]]. This "constellation" consists of Thr-166, Arg-162, and Glu-184, which is similar to the catalytic triad residues of the serine protease [[trypsin]], which are Ser-195, His-57, Asp-102 <ref> PMID:12475199</ref>.

Threonine and Glutamic acid are found in other catalytic triads of the serine hydrolase family, but argenine seems unlikely to replace histidine as a base because of its higher pKa. There have been studies that have shown that Argenine is essential for other enzymatic functions, such as in the Ser-Arg-Asp triad in cytosolic phospholipase A2 and as a catalytic base in Sortase A. <scene name='User:Olivia_Cheng/Sandbox_1/1p4p_asn164/1'>Asn-164</scene> forms an H-bond with <scene name='User:Olivia_Cheng/Sandbox_1/1p4p_asn164/1'>Thr-166</scene> and may rearrange to form a putative oxyanion hole with Thr-166 and another unidentified atom if active in the catalysis. A concerted proton transfer, similar to a “proton wire”, is one plausible mechanism that would allow argenine to function in the catalytic triad of a protease <ref name="Structural"/>.

== Potential Oxidative Mechanism ==
It was recently discovered that all antibodies contained Fab portions that catalyzed a reaction between singlet oxygen and water, yielding hydrogen peroxide, ozone, water and hydroxide radicals. Hydrogen peroxide is a toxic oxidative species and might be the product of an ancient mechanism to protect against infection. UV absorption increases the rate for this reaction. B. burgdorferi is especially vulnerable to oxidative damage because its ecological niche is in areas with limited oxygen and its genome does not encode a catalase. This oxidative mechanism might explain why some mABs are bactericidal without the use of complement <ref name="Structural"/>.

= 3D Structures =

[[Monoclonal_Antibody]]
 
[[Trypsin]]

= Links =

*[cdc.gov/lyme] - the Official Lyme Disease resource of the US Center for Disease Control.

= References =

<references />

User:Olivia Cheng/Sandbox 1

2012-05-04T20:44:00Z

Olivia Cheng:

Outer Surface Protein B (OspB) has been found to play a vital role in the adherence of B. bugdorferi onto tick guts, which promote the survival of the vector and spread of Lyme disease. OspB-deficient B. budgdorferi have been found to bind poorly to tick gut extracts <ref>PMID: 17352535 </ref>.

= Background =
<Structure load='1rjl' size='450' frame='true' align='right' caption='OspB interacting with Fab H6831' scene='User:Olivia_Cheng/Sandbox_1/1rjl_color/1' />

Lyme disease, discovered in 1975, is spread by means of a tick vector that carries the causative agent, spirochete [http://en.wikipedia.org/wiki/Borrelia_burgdorferi ''Borrelia Burgdorferi''] <ref name="Structural">PMID:15864264</ref>. It is the most frequently reported vectorborne illness in the United States. Individuals who have this disease may show symptoms including skin lesions called erythema migans and the characteristic bulls-eye rash <ref>http://www.cdc.gov/lyme/stats/index.html</ref>. OspA and OspB are the major outer surface proteins found on ''B. Borrelia''. OspB has shown significant variability in amino acid sequence and antigen reactivity in comparison to OspA, known to be largely invariant <ref name="Versatile">PMID:15713683</ref>.

= OspB Interaction with Fab of H6831 =
The [http://en.wikipedia.org/wiki/Lyme_disease_microbiology#Outer_surface_proteins outer-surface proteins] (Osps) in ''B. burgdorferi'' spirochete activate the classical and alternative pathways of the complement system. B. burgdorferi is resistant to complement mediated lysis. The complement inhibitor factor H binds to Osps and the C3b cascade is deactivated <ref name="Versatile"/>.

Within this complex is <scene name='User:Olivia_Cheng/Sandbox_1/1rjl_chainc_transparentabd/6'>a truncated form of OspB</scene>, shown in purple. The H6831 Fab is shown in white. (Click <scene name='User:Olivia_Cheng/Sandbox_1/1rjl_color/1'>here</scene> to revert back to the original.)

H6831 is an IgG class monoclonal complement-independent antibody shown to effectively lyse outer surface protein B (OspB) of ''B. burgdorferi''. H6831 recognizes <scene name='User:Olivia_Cheng/Sandbox_1/1rjl_chainc_lys253/6'>Lys-253</scene> on OspB. Studies have shown that ''B. burgdorferi'' strains with Thr, Cys, Gly, or Glu instead of Lys decrease the binding affinity between H6831 and OspB. The sequence and structure of bactericidal H6831 Fab are typical for IgG2 heavy chain/kappa light chain class. <ref name="Versatile"/>.

The H6831 epitope of OspB is topologically equivalent to LA-2 epitope of OspA <ref name="Versatile"/>. Similar to the LA-2 epitope, the H6831 epitope is positioned opposite the N-terminus near the end of the antigen. The buried surface area of OspB in the H6831 Fab complex is smaller than that of the OspA-LA2 complex. Loop 1 in the OspA-LA2 complex has the most interactions with the Fab, where as Loop 1 in the OspB-H6831 complex has the fewest interactions with the Fab <ref>PMID: 9038292 </ref>.

== Binding ==
<Structure load='1P4P' size='250' frame='true' align='right' caption='Proteolysed Portion of OspB' scene='User:Stephanie_Maung/Sandbox/Gray_1p4p/1' />

H6831 Fab complex binds to a highly accessible region near the C-terminus of OspB, away from the N-terminus lipid anchor. The interaction of OspB-Fab complex depends heavily on hydrogen bonding between <scene name='User:Stephanie_Maung/Sandbox/1p4p_proteolysed_region_loops/5'>loops 1, 2, and 3</scene>. When binding of full-length OspB to Fab fragments of H6831 or CB2 fail, it is usually because <scene name='User:Stephanie_Maung/Sandbox/1p4p_proteolysed_region_loops/3'>Lys-253</scene>, located on loop 2, has been replaced with a different residue. Studies show that substitutions in basic residues of hen egg-white lysozyme (HEL) that participate in HEL-Fab complexes decreased binding affinity by 400-10,000 times. <ref name="Versatile"/>

Due to its effective bactericidal actions, H6831 is used to generate less virulent escape variants of ''B. burgdorferi'' <ref name="Versatile"/>. In the majority of the mutations created from in vivo and in vitro immunization of mice, truncated forms of OspB within the C terminus lead to premature stop codons<ref>PMID:8308101 </ref>. It has been suggested that OspB mutants are more sensitive to proteolysis due to missense mutations that disturb the conformation of OspB <ref name="Versatile"/>. Truncated OspBs cease within the two C-terminal beta-strands of the central sheet. H6831 disorders or removes a beta sheet from OspB after binding. Cleavage may be a possible explanation for the conformational changes of OspB <ref>PMID:9125579</ref>. In <scene name='User:Stephanie_Maung/Sandbox/Gray_1p4p/1'>H-6831 free</scene> and <scene name='User:Stephanie_Maung/Sandbox/1rjl_chainc/1'>H-6831 bound</scene> forms of OspB, some changes result from proteolysis near the N terminus <ref name="Versatile"/>. Residues 157 - 201 on OspB contain the <scene name='User:Stephanie_Maung/Sandbox/1p4p_proteolysed_region_triad/2'>cleaved region</scene>, shown in plum.

Aromatic residues tyrosine and tryptophan are also present in the OspB-H6831 interaction, a feature found in many antigen-antibody complexes. The Lys-253 residue forms a trans conformation between these aromatic residues of H6831. In the complex structure of the antibody binding site, the electron density is well defined and shows increased contact between Lys-253 and the antigen-binding site of the Fab. Most of the electrostatic and hydrogen-bond interactions occur between loop 2 and the Fab heavy chain <ref name="Structural"/>.

= Potential Mechanisms of Lysis =

== Potential Catalytic Triad ==

<Structure load='1p4p' size='250' frame='true' align='left' caption='OspB' scene='Insert optional scene name here' />

[[Image: Cataly.png|300px|right|thumb| Comparison of catalytic triads]]

The mechanism by which H6831 Fab destroys a spirochete appears to be a novel interaction. It is possible that Fab binding changes the properties of OspB folding, which may increase sensitivity of the protein to proteolysis or aggregation. NMR methods showed that the effects of binding can be sent to regions of the antigen distant from epitope, which is at the <scene name='User:Olivia_Cheng/Sandbox_1/1p4p_rainbow/1'>C-terminus</scene> shown in blue (N-terminus in red). OspB shows signs of truncation after interacting with Fab of H6831 <ref>PMID: 1382591</ref>.

It is possible that OspB performs an autoproteolysis. There is a set of three residues found on OspB that resembles the catalytic triad of Serine_Proteases. This "constellation" consists of Thr-166, Arg-162, and Glu-184, which is similar to the catalytic triad residues of the serine protease trypsin, which are Ser-195, His-57, Asp-102 <ref> PMID:12475199</ref>.

Threonine and Glutamic acid are found in other catalytic triads of the serine hydrolase family, but argenine seems unlikely to replace histidine as a base because of its higher pKa. There have been studies that have shown that Argenine is essential for other enzymatic functions, such as in the Ser-Arg-Asp triad in cytosolic phospholipase A2 and as a catalytic base in Sortase A. Asn-164 forms an H-bond with Thr-166 and may rearrange to form a putative oxyanion hole with Thr-166 and another unidentified atom if active in the catalysis <ref name="Structural"/>.

== Potential Oxidative Mechanism ==
It was recently discovered that all antibodies contained Fab portions that catalyzed a reaction between singlet oxygen and water, yielding hydrogen peroxide, ozone, water and hydroxide radicals. Hydrogen peroxide is a toxic oxidative species and might be the product of an ancient mechanism to protect against infection. UV absorption increases the rate for this reaction. B. burgdorferi is especially vulnerable to oxidative damage because its ecological niche is in areas with limited oxygen and its genome does not encode a catalase. This oxidative mechanism might explain why some mABs are bactericidal without the use of complement. <ref name="Structural"/>.

= 3D Structures =

[[Monoclonal_Antibody]]
 
[[Trypsin]]

= Links =

*[cdc.gov/lyme] - the Official Lyme Disease resource of the US Center for Disease Control.

= References =

<references />

<scene name='User:Olivia_Cheng/Sandbox_1/1rjl_color/1'>reg</scene>

<scene name='User:Olivia_Cheng/Sandbox_1/1rjl_chainc_lys253/6'>lys on 1rjl</scene>

<scene name='User:Olivia_Cheng/Sandbox_1/1rjl_chainc_transparentabd/6'>transparent spin</scene>

<scene name='User:Olivia_Cheng/Sandbox_1/1rjl_chainc/7'>1rjl chain c</scene>

<Structure load='1rjl' size='500' frame='true' align='right' caption='Insert caption here' scene='User:Olivia_Cheng/Sandbox_1/1rjl_color/1' />

<scene name='User:Olivia_Cheng/Sandbox_1/1p4p_activesite/2'>1p4p</scene>

<scene name='User:Olivia_Cheng/Sandbox_1/1p4p_cat_triad_cool/9'>cat triad</scene>

==

H6831 is a monoclonal complement-independent antibody to outer surface protein B (OspB).

[[Serine_Proteases]]

[[trypsin]]

User:Olivia Cheng/Sandbox 1

2012-05-04T20:34:34Z

Olivia Cheng:

Outer Surface Protein B (OspB) has been found to play a vital role in the adherence of B. bugdorferi onto tick guts, which promote the survival of the vector and spread of Lyme disease. OspB-deficient B. budgdorferi have been found to bind poorly to tick gut extracts <ref>PMID: 17352535 </ref>.

= Background =
<Structure load='1rjl' size='450' frame='true' align='right' caption='OspB interacting with Fab H6831' scene='User:Olivia_Cheng/Sandbox_1/1rjl_color/1' />

Lyme disease, discovered in 1975, is spread by means of a tick vector that carries the causative agent, spirochete [http://en.wikipedia.org/wiki/Borrelia_burgdorferi ''Borrelia Burgdorferi''] <ref name="Structural">PMID:15864264</ref>. It is the most frequently reported vectorborne illness in the United States. Individuals who have this disease may show symptoms including skin lesions called erythema migans and the characteristic bulls-eye rash <ref>http://www.cdc.gov/lyme/stats/index.html</ref>. OspA and OspB are the major outer surface proteins found on ''B. Borrelia''. OspB has shown significant variability in amino acid sequence and antigen reactivity in comparison to OspA, known to be largely invariant <ref name="Versatile">PMID:15713683</ref>.

= OspB Interaction with Fab of H6831 =
The [http://en.wikipedia.org/wiki/Lyme_disease_microbiology#Outer_surface_proteins outer-surface proteins] (Osps) in ''B. burgdorferi'' spirochete activate the classical and alternative pathways of the complement system. B. burgdorferi is resistant to complement mediated lysis. The complement inhibitor factor H binds to Osps and the C3b cascade is deactivated <ref name="Versatile"/>.

Within this complex is <scene name='User:Olivia_Cheng/Sandbox_1/1rjl_chainc_transparentabd/6'>a truncated form of OspB</scene>, shown in purple. The H6831 Fab is shown in white. (Click <scene name='User:Olivia_Cheng/Sandbox_1/1rjl_color/1'>here</scene> to revert back to the original.)

H6831 is an IgG class monoclonal complement-independent antibody shown to effectively lyse outer surface protein B (OspB) of ''B. burgdorferi''. H6831 recognizes <scene name='User:Olivia_Cheng/Sandbox_1/1rjl_chainc_lys253/6'>Lys-253</scene> on OspB. Studies have shown that ''B. burgdorferi'' strains with Thr, Cys, Gly, or Glu instead of Lys decrease the binding affinity between H6831 and OspB. The sequence and structure of bactericidal H6831 Fab are typical for IgG2 heavy chain/kappa light chain class. <ref name="Versatile"/>.

The H6831 epitope of OspB is topologically equivalent to LA-2 epitope of OspA <ref name="Versatile"/>. Similar to the LA-2 epitope, the H6831 epitope is positioned opposite the N-terminus near the end of the antigen. The buried surface area of OspB in the H6831 Fab complex is smaller than that of the OspA-LA2 complex. Loop 1 in the OspA-LA2 complex has the most interactions with the Fab, where as Loop 1 in the OspB-H6831 complex has the fewest interactions with the Fab <ref>PMID: 9038292 </ref>.

== Binding ==
<Structure load='1P4P' size='250' frame='true' align='right' caption='Proteolysed Portion of OspB' scene='User:Stephanie_Maung/Sandbox/Gray_1p4p/1' />

H6831 Fab complex binds to a highly accessible region near the C-terminus of OspB, away from the N-terminus lipid anchor. The interaction of OspB-Fab complex depends heavily on hydrogen bonding between <scene name='User:Stephanie_Maung/Sandbox/1p4p_proteolysed_region_loops/5'>loops 1, 2, and 3</scene>. When binding of full-length OspB to Fab fragments of H6831 or CB2 fail, it is usually because <scene name='User:Stephanie_Maung/Sandbox/1p4p_proteolysed_region_loops/3'>Lys-253</scene>, located on loop 2, has been replaced with a different residue. Studies show that substitutions in basic residues of hen egg-white lysozyme (HEL) that participate in HEL-Fab complexes decreased binding affinity by 400-10,000 times. <ref name="Versatile"/>

Due to its effective bactericidal actions, H6831 is used to generate less virulent escape variants of ''B. burgdorferi'' <ref name="Versatile"/>. In the majority of the mutations created from in vivo and in vitro immunization of mice, truncated forms of OspB within the C terminus lead to premature stop codons<ref>PMID:8308101 </ref>. It has been suggested that OspB mutants are more sensitive to proteolysis due to missense mutations that disturb the conformation of OspB <ref name="Versatile"/>. Truncated OspBs cease within the two C-terminal beta-strands of the central sheet. H6831 disorders or removes a beta sheet from OspB after binding. Cleavage may be a possible explanation for the conformational changes of OspB <ref>PMID:9125579</ref>. In <scene name='User:Stephanie_Maung/Sandbox/Gray_1p4p/1'>H-6831 free</scene> and <scene name='User:Stephanie_Maung/Sandbox/1rjl_chainc/1'>H-6831 bound</scene> forms of OspB, some changes result from proteolysis near the N terminus <ref name="Versatile"/>. Residues 157 - 201 on OspB contain the <scene name='User:Stephanie_Maung/Sandbox/1p4p_proteolysed_region_triad/2'>cleaved region</scene>, shown in plum.

Aromatic residues tyrosine and tryptophan are also present in the OspB-H6831 interaction, a feature found in many antigen-antibody complexes. The Lys-253 residue forms a trans conformation between these aromatic residues of H6831. In the complex structure of the antibody binding site, the electron density is well defined and shows increased contact between Lys-253 and the antigen-binding site of the Fab. Most of the electrostatic and hydrogen-bond interactions occur between loop 2 and the Fab heavy chain <ref name="Versatile"/>

= Potential Mechanisms of Lysis =

== Potential Catalytic Triad ==

<Structure load='1p4p' size='250' frame='true' align='left' caption='OspB' scene='Insert optional scene name here' />

[[Image: Cataly.png|300px|right|thumb| Comparison of catalytic triads]]

The mechanism by which H6831 Fab destroys a spirochete appears to be a novel interaction. It is possible that Fab binding changes the properties of OspB folding, which may increase sensitivity of the protein to proteolysis or aggregation. NMR methods showed that the effects of binding can be propagated to regions of the antigen that are distant from epitope.” OspB shows signs of truncation after interacting with Fab of H6831 <ref>PMID: 1382591</ref>.

It is possible that OspB performs an autoproteolysis. There is a <scene name='User:Olivia_Cheng/Sandbox_1/1p4p_cat_triad_cool/9'>set of three residues</scene> found on OspB that resembles the catalytic triad of [[Serine_Proteases]]. This "constellation" consists of Thr-166, Arg-162, and Glu-184, which is similar to the catalytic triad residues of the serine protease [[trypsin]], which are Ser-195, His-57, Asp-102 <ref> PMID:12475199</ref>.

Threonine and Glutamic acid are found in other catalytic triads of the serine hydrolase family, but argenine seems unlikely to replace histidine as a base because of its higher pKa. There have been studies that have shown that Argenine is essential for other enzymatic functions, such as in the Ser-Arg-Asp triad in cytosolic phospholipase A2 and as a catalytic base in Sortase A. <scene name='User:Olivia_Cheng/Sandbox_1/1p4p_asn164/1'>Asn-164</scene> forms an H-bond with Thr-166 and may rearrange to form a putative oxyanion hole with Thr-166 and another unidentified atom if active in the catalysis. A concerted proton transfer, similar to a “proton wire”, is one plausible mechanism that would allow argenine to function in the catalytic triad of a protease <ref name=”Structural”/>.

== Potential Oxidative Mechanism ==
It was recently discovered that all antibodies contained Fab portions that catalyze a reaction between singlet oxygen and water, which yields hydrogen peroxide, ozone, water and hydroxide radicals. Hydrogen peroxide is a toxic oxidative species and is the product of a possible ancient mechanism used protect against infection. UV absorption increases the rate for this reaction. B. burgdorferi is especially vulnerable to oxidative damage because its ecological niche is areas with limited oxygen and its genome does not encode a catalase. This oxidative mechanism might explain why some mABs are bactericidal without the use of complement <ref name=”Structural”/>.

= 3D Structures =

[[Monoclonal_Antibody]]
 
[[Trypsin]]

= Links =

*[cdc.gov/lyme] - the Official Lyme Disease resource of the US Center for Disease Control.

= References =

<references />

<scene name='User:Olivia_Cheng/Sandbox_1/1rjl_color/1'>reg</scene>

<scene name='User:Olivia_Cheng/Sandbox_1/1rjl_chainc_lys253/6'>lys on 1rjl</scene>

<scene name='User:Olivia_Cheng/Sandbox_1/1rjl_chainc_transparentabd/6'>transparent spin</scene>

<scene name='User:Olivia_Cheng/Sandbox_1/1rjl_chainc/7'>1rjl chain c</scene>

<Structure load='1rjl' size='500' frame='true' align='right' caption='Insert caption here' scene='User:Olivia_Cheng/Sandbox_1/1rjl_color/1' />

<scene name='User:Olivia_Cheng/Sandbox_1/1p4p_activesite/2'>1p4p</scene>

<scene name='User:Olivia_Cheng/Sandbox_1/1p4p_cat_triad_cool/9'>cat triad</scene>

==

H6831 is a monoclonal complement-independent antibody to outer surface protein B (OspB).

[[Serine_Proteases]]

[[trypsin]]

File:Cataly.png

2012-05-03T15:22:48Z

Olivia Cheng: Comparison between catalytic triad of trypsin and potential catalytic triad of OspB.

Comparison between catalytic triad of trypsin and potential catalytic triad of OspB.

File:Untitled.png

2012-05-03T15:21:50Z

Olivia Cheng: uploaded a new version of "Image:Untitled.png": Reverted to version as of 18:47, 30 April 2012

File:Untitled.png

2012-05-03T15:21:32Z

Olivia Cheng: uploaded a new version of "Image:Untitled.png": Comparison between catalytic triad of trypsin and potential catalytic triad of OspB.