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{{Sandbox_ESBS_2012}}
{{Sandbox_ESBS_2012}}
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Please, do not delete or modify text or images, this page is reserved for a work from two students in ESBS. Thank you.




Please, do not delete or modify text or images, this page is reserved for a work from two students in ESBS. Thank you.
<big>'''Crystal structure of the small GTPase Rab27B complexed with the Slp homology domain of Melanophilin'''</big>
 
[[Image:2zet.png|left|200px]] {{STRUCTURE_2zet|  PDB=2zet  |  SCENE=  }}






<big>'''Crystal structure of the small GTPase Rab27B complexed with the Slp homology domain of Melanophilin'''</big>
=='''Introduction'''==
=='''Introduction'''==


The Rab family of proteins is one of the Ras family of monomeric G proteins. There are approximately 70 types of known Rabs in humans. Rab GTPases are involved in the regulation of vesicular traffic in eukaryotic cells, among which vesicle formation, vesicle movement along actin and tubulin network and membrane fusion. Rab proteins are bound at the surface of the membrane by a lipid group covalently linked to an amino acid. As Rabs are GTPases, they have two conformations. The inactive form is bound to GDP and the active one is linked to GTP. Rab escort proteins only bound Rab-GDP and Rab effectors only bound Rab-GTP. Rabs work thanks to Rab effectors.
The Rab family of proteins is one of the Ras family of monomeric G proteins. There are approximately 70 types of known Rabs in humans. Rab GTPases are involved in the regulation of vesicular traffic in eukaryotic cells, among which vesicle formation, vesicle movement along actin and tubulin network and membrane fusion. Rab proteins are bound to the surface of the membrane by a lipid group covalently linked to an amino acid. As Rabs are GTPases, they have two conformations. The inactive form is bound to GDP and the active one is linked to GTP. Rab escort proteins only bind Rab-GDP and Rab effectors only bind Rab-GTP. Rabs work thanks to Rab effectors.


[[Image:Cycle_of_the_small_G_protein_Rab.jpg |thumb|frame|Cycle of the small G protein Rab]]<ref>http://www.ncbi.nlm.nih.gov/pmc/articles/PMC138937/</ref>
[[Image:Cycle_of_the_small_G_protein_Rab.jpg |thumb|frame| Figure1: Cycle of the small G protein Rab <ref>http://www.ncbi.nlm.nih.gov/pmc/articles/PMC138937/</ref>]]


Rab27A and Rab27B are isoforms (72% of identity) and use several specific effector proteins, among which the Exophilin3/Melanophilin/Slac2-a, to regulate the exocytosis of secretory granule cells. Two groups of Rab27 effectors can be made, depending on their interactions specificities. The first one is composed of Melanophilin, Exophilin4, Exophilin5 and Exophilin6, which are specific effectors for Rab27. The other group consists of Rabphilin-3a, Granuphilin-a and JFC1, which are also effectors for Rab3 and Rab8 for instance. Rab27A/B proteins are not only involved in the transport of lysosome-related organelles regulation and may regulate more types of granule exocytosis mechanisms, like endocrine.<ref>PMID:18940604</ref>
Rab27A and Rab27B are isoforms (72% of identity) and use several specific effector proteins, among which the Exophilin3/Melanophilin/Slac2-a, to regulate the exocytosis of secretory granule cells. Two groups of Rab27 effectors can be made, depending on their interactions specificities. The first one is composed of Melanophilin, Exophilin4, Exophilin5 and Exophilin6, which are specific effectors for Rab27. The other group consists of Rabphilin-3a, Granuphilin-a and JFC1, which are also effectors for Rab3 and Rab8 for instance. Rab27A/B proteins are not only involved in the transport of lysosome-related organelles regulation and may regulate more types of granule exocytosis mechanisms, like endocrine.<ref>PMID:18940604</ref>


Mutations of Rab27A cause human type II Griscelli syndrome (hypopigmentation and immunodeficiency disorder) because of a defect in melanosome transport in melanocytes. Rab27A orchestrates the transport of melanosomes by recruitment of the actin motor, myosin Va, onto melanosomes. Rab27A links the melanosome and then recruits melanophilin, which finally bounds myosin-Va. Because Rab27A and Rab27B are isoforms and the effects of Rab27A mutations, it is interested to study Rab27B.<ref>http://www.spring8.or.jp/pdf/en/res_fro/08/028-029.pdf</ref>
Mutations of Rab27A cause human type II Griscelli syndrome (hypopigmentation and immunodeficiency disorder) because of a defect in melanosome transport in melanocytes. Rab27A orchestrates the transport of melanosomes by recruitment of the actin motor, myosin Va, onto melanosomes. Rab27A links the melanosome and then recruits melanophilin, which finally bounds myosin-Va. Because Rab27A and Rab27B are isoforms and because of the effects of Rab27A mutations, it is interested to study Rab27B.<ref>http://www.spring8.or.jp/pdf/en/res_fro/08/028-029.pdf</ref>
 
 


=='''Biological functions'''==
=='''Biological functions'''==
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==='''Rab27B'''===
==='''Rab27B'''===


At the moment, no Rab27B mutation is known to cause human disease or animal strain. But Rab27B is known to localize on pituitary endocrines granules, dense and α-granules in platelets and megakaryocytes, urothelial fusiform vesicles and parotid and pancreatic acinar granules. Rab27B is largely expressed in canonical secretory cells, neurons and cells involved in surface protection and mechanical extension. Rab27B regulates a secretory granule exocytosis step in parotid acinar cells. It has several Rab27B effector proteins. For example, the interaction of Rab27B with Slac2-c/MyRIP is very important for amylase release. Slac2-c is a myosin Va/VIIa and actin binding protein and may work for retinal melanosome transport in melanocytes regulation. Rab27B exists on amylase containing secretory granules in the rat parotid gland. That is why the Rab27B-Slac2-c complex is important to release amylase. Rab27B is the first Rab protein known to contribute in the exocytosis of secretory granules in parotid acinar cells.<ref>http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2043558/</ref>
At the moment, no Rab27B mutation is known to cause human disease or animal strain. But Rab27B is known to localize on pituitary endocrines granules, dense and α-granules in platelets and megakaryocytes, urothelial fusiform vesicles and parotid and pancreatic acinar granules. Rab27B is largely expressed in canonical secretory cells, neurons and cells involved in surface protection and mechanical extension. Rab27B regulates a secretory granule exocytosis step in parotid acinar cells. It has several Rab27B effector proteins. For example, the interaction of Rab27B with Slac2-c/MyRIP is very important for amylase release. Slac2-c is a myosin Va/VIIa and actin binding protein and may act in retinal melanosome transport in melanocytes regulation. Rab27B exists on amylase containing secretory granules in the rat parotid gland. That is why the Rab27B-Slac2-c complex is important to release amylase. Rab27B is the first Rab protein known to contribute in the exocytosis of secretory granules in parotid acinar cells.<ref>http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2043558/</ref>


==='''Exophilin3/Slac2-a/Melanophilin'''===
==='''Exophilin3/Slac2-a/Melanophilin'''===
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== '''Structure''' ==
== '''Structures''' ==


2ZET is a 4 chains structure of sequences from [http://en.wikipedia.org/wiki/Mus_musculus Mus musculus]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=2ZET OCA].
2ZET is a 4 chains structure of sequences from [http://en.wikipedia.org/wiki/Mus_musculus Mus musculus]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=2ZET OCA].


=== '''Structure of Rab27B''' ===
=== '''Structure of Rab27B''' ===
 
[[Image:Rab27B slac2a image1.jpg | 425 px | thumb | right |Figure 2: Overall stucture of the Rab27B/Slac2-a complex. <ref>PMID:18940604</ref>]]
[[Image:Rab27B slac2a image2.jpg | 500 px | thumb | right |Picture 2:   Caption: ]]


'''General informations:'''  
'''General informations:'''  
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Rab27B adopts a globular form in the complex. The  
Rab27B adopts a globular form in the complex. The  
<scene name='Sandbox_Reserved_706/Loop_entre_beta_2_et_3/1'>ß2 and ß3 loop (residues 55-64)</scene> and <scene name='Sandbox_Reserved_706/Rab27b_loop_2_alpha3_b5/1'>α3-ß5 loop (residues122-126)</scene> of Rab27B are both regions involved in Slac2-a binding. <ref>PMID:18940604</ref>
<scene name='Sandbox_Reserved_706/Loop_entre_beta_2_et_3/1'>ß2 and ß3 loop (residues 55-64)</scene> and <scene name='Sandbox_Reserved_706/Rab27b_loop_2_alpha3_b5/1'>α3-ß5 loop (residues122-126)</scene> of Rab27B are both regions involved in Slac2-a binding. <ref>PMID:18940604</ref>
[[Image:Rab27B slac2a image2.jpg | 450 px | thumb | left |Figure 3: Structural comparaison of Rab27B in GTP and GDP form. <ref>PMID:18940604</ref>]]


[[Image:Rab27B slac2a image1.jpg | 425 px | thumb | left |Picture 1:  Caption: ]]
The complex Rab27B/Slac2-a can only be formed when Rab27B is in a GTP form. This can be explained because of the changing of shape between Rab27B in the GTP and GDP form (see figure 3).
The complex Rab27B/Slac2-a can only be formed when Rab27B is in a GTP form. This can be explained because of the changing of shape between Rab27B in the GTP and GDP form. (See figure 3)
Rab27B–GTP adopts a compact monomer conformation, while an extended conformation is observed for GDP-bound Rab27B.
Rab27B–GTP adopts a compact monomer conformation, while an extended conformation is observed for GDP-bound Rab27B.
<scene name='Sandbox_Reserved_706/Switch_1_rab27b/1'>Switch 1</scene> and <scene name='Sandbox_Reserved_706/Switch2_rab27b/1'>switch 2</scene> of Rab27B play an important role in this changing of conformation. In fact, switch 1 and 2 allow GTP binding by making hydrogen bond with GTP's γ-phosphate, and this way also allows Slac2-a binding. But in the complex Rab27B/GDP switch 1 and switch 2 exist completely apart from the bound GDP, so there is no interaction with Slac2-a and no binding.
<scene name='Sandbox_Reserved_706/Switch_1_rab27b/1'>Switch 1</scene> and <scene name='Sandbox_Reserved_706/Switch2_rab27b/1'>switch 2</scene> of Rab27B play an important role in this changing of conformation. In fact, switch 1 and 2 allow GTP binding by making hydrogen bond with GTP's γ-phosphate, and this way also allows Slac2-a binding. But in the complex Rab27B/GDP switch 1 and switch 2 exist completely apart from the bound GDP, so there is no interaction with Slac2-a and no binding.


=== '''Structure of Slac2-a''' ===
=== '''Structure of Slac2-a''' ===
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The structure of the Slac2-a effector domain comprises three subdomains: SHD1, SHD2, and a zinc-binding subdomain that is flanked by the two SHDs. <ref>PMID:11856727</ref>  
The structure of the Slac2-a effector domain comprises three subdomains: SHD1, SHD2, and a zinc-binding subdomain that is flanked by the two SHDs. <ref>PMID:11856727</ref>  
SHD1 is folded into a 66 Å <scene name='Sandbox_Reserved_706/Helice_1_de_slac2a/1'>lond helix alpha 1</scene>, and SHD2 is composed of <scene name='Sandbox_Reserved_706/Alpha_3_5_shd2/3'>three alpha helices, alpha 3 to alpha 5</scene>. SHD1 and SHD2 interact with each other by making a coiled coil between α1 and α3–α5. In this coiled coil, α3 and α5 interact with α1 in an antiparallel manner (see figure 2).  
SHD1 is folded into a 66 Å <scene name='Sandbox_Reserved_706/Helice_1_de_slac2a/1'>lond helix alpha 1</scene>, and SHD2 is composed of <scene name='Sandbox_Reserved_706/Alpha_3_5_shd2/3'>three alpha helices, alpha 3 to alpha 5</scene>. SHD1 and SHD2 interact with each other by making a coiled coil between α1 and α3–α5. In this coiled coil, α3 and α5 interact with α1 in an antiparallel manner (see figure 2).  
The <scene name='Sandbox_Reserved_706/Zinc_binding_domain_slac2a/1'>zinc-binding subdomain</scene> contains four short {{Template:ColorKey_Strand}} (ß1–ß4) and five loops(L1-L5), including one short {{Template:ColorKey_Helix}} α2 and three short helices (in pupule). The <scene name='Sandbox_Reserved_706/Zn1_et_zn2_de_slac2a/1'>two zinc ions (Zn1 and Zn2)</scene> are each coordinated by four conserved cysteine residues:
The <scene name='Sandbox_Reserved_706/Zinc_binding_domain_slac2a/1'>zinc-binding subdomain</scene> contains four short {{Template:ColorKey_Strand}} (ß1–ß4) and five loops(L1-L5), including one short {{Template:ColorKey_Helix}} α2 and three short helices (in purple). The <scene name='Sandbox_Reserved_706/Zn1_et_zn2_de_slac2a/1'>two zinc ions (Zn1 and Zn2)</scene> are each coordinated by four conserved cysteine residues:
<scene name='Sandbox_Reserved_706/Cys_64_67_89_92/2'>Cys64, Cys67, Cys89 and Cys92</scene> for Zn1 binding, and <scene name='Sandbox_Reserved_706/Cys81_cys84_cys104_cys107/2'>Cys81, Cys84, Cys104, and Cys107</scene> for Zn2 binding. <ref>PMID:18940604</ref>
<scene name='Sandbox_Reserved_706/Cys_64_67_89_92/2'>Cys64, Cys67, Cys89 and Cys92</scene> for Zn1 binding, and <scene name='Sandbox_Reserved_706/Cys81_cys84_cys104_cys107/2'>Cys81, Cys84, Cys104, and Cys107</scene> for Zn2 binding. <ref>PMID:18940604</ref>


 
<Structure load='2ZET' size='500' frame='true' align='right' caption='Final model of 2zet: Two Rab27B/Slac2-a complexes (A/C and B/D)' scene='Insert optional scene name here' />
 
 
<Structure load='2ZET' size='500' frame='true' align='right' caption='Final model of 2zet: Two Rab27B/Slac2-a complexes (A/C and B/D) (' scene='Insert optional scene name here' />


=== '''Structure of the complex''' ===
=== '''Structure of the complex''' ===
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Rab27B interacts with Slac2-a with a buried surface area of 2534 Ų, which corresponds to 30.2% of the total surface area.
Rab27B interacts with Slac2-a with a buried surface area of 2534 Ų, which corresponds to 30.2% of the total surface area.
Rab27B binds to Slac2-a with good shape complementarity and this interaction involves many hydrophobic interactions and several electrostatic interactions.
 
There are three contact areas in the interface between Rab27B and Slac2-a.  The first and second contact areas involve the coiled-coil regions of Slac2-a and constitute the main interface.
There are three contact areas in the interface between Rab27B and Slac2-a.  The first and second contact areas involve the coiled-coil regions of Slac2-a and constitute the main interface.
[[Image:Rab27B slac2a image3.gif | 200 px | thumb | left |Picture 4: Rab27B/Slac2-a contact interfaces :
 
[[Image:Rab27B slac2a image3.gif | 225 px | thumb | left |Figure 4: Rab27B/Slac2-a contact interfaces :
(A) Contact area 1,  
(A) Contact area 1,  
(C) Contact area 2,  
(C) Contact area 2,  
(E) Contact area 3.]]
(E) Contact area 3.<ref>PMID:18940604</ref>]]


*The first contact area of the Rab27B/Slac2-a interface consists of <scene name='Sandbox_Reserved_706/6a9_rabcdr/2'> residues 6 to 9 from  the Rab complementarity-determining regions (RabCDR)</scene> , part of the switch and interswitch regions of Rab27B, and the coiled-coil regions of Slac2-a.<ref>PMID:18940604</ref> In this first contact area we can find three different types of interactions, electrostatic, hydrophobic and hydrogen bonds: (see figure 4A)
*The first contact area of the Rab27B/Slac2-a interface consists of <scene name='Sandbox_Reserved_706/6a9_rabcdr/2'> residues 6 to 9 from  the Rab complementarity-determining regions (RabCDR)</scene> , part of the switch and interswitch regions of Rab27B, and the coiled-coil regions of Slac2-a.<ref>PMID:18940604</ref> In this first contact area we can find three different types of interactions, electrostatic, hydrophobic and hydrogen bonds: (see figure 4A)
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At this interface, there are hydrophobic interactions and three hydrogen bonds.
At this interface, there are hydrophobic interactions and three hydrogen bonds.
<scene name='Sandbox_Reserved_706/Tyr121_arg90/1'>Tyr121 of Slac2-a  form a hydrogen bond with the Arg90</scene> carbonyl group of Rab27B.
<scene name='Sandbox_Reserved_706/Tyr121_arg90/1'>Tyr121 of Slac2-a  form a hydrogen bond with the Arg90</scene> carbonyl group of Rab27B.
Arg128 of Slac2-a, which is highly conserved among the Slp-family proteins, makes two hydrogen bonds with the carbonyl groups of Gln118 and Ala121 of Rab27B
<scene name='Sandbox_Reserved_706/Arg128_ala121gln118/1'>Arg128 of Slac2-a, which is highly conserved among the Slp-family proteins, makes two hydrogen bonds with the carbonyl groups of Gln118 and Ala121 of Rab27B</scene>.
* The third contact area is the smallest one . It  involves the zinc-binding subdomain of Slac2-a and the b2–b3 loop of Rab27B, with superposition of their molecular surfaces. (See figure 4E)
* The third contact area is the smallest one . It  involves the <scene name='Sandbox_Reserved_706/Zinc_binding_domain_slac2a/1'>zinc-binding subdomain</scene> of Slac2-a and the <scene name='Sandbox_Reserved_706/Loop_entre_beta_2_et_3/1'>β2–β3 loop</scene> of Rab27B, with superposition of their molecular surfaces (see figure 4E).<ref>PMID:18940604</ref>
 




These findings strongly suggested that Tyr6 from RabCDR and three residues (Leu84, Phe88, and Asp91) from switch 2 of Rab27 are the minimum determinants of Slac2-a binding.<ref>PMID:18940604</ref>
These findings strongly suggested that <scene name='Sandbox_Reserved_706/Leu84_phe88_asp91_tyr6/1'>Tyr6 from RabCDR and three residues, Leu84, Phe88, and Asp91</scene> from switch 2 of Rab27 are the minimum determinants of Slac2-a binding.<ref>PMID:18940604</ref>


== '''External Resources''' ==
== '''External Resources''' ==
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* [http://en.wikipedia.org/wiki/Mus_musculus Mus musculus]
* [http://en.wikipedia.org/wiki/Mus_musculus Mus musculus]
* [http://oca.weizmann.ac.il/oca-bin/ocashort?id=2ZET OCA]
* [http://oca.weizmann.ac.il/oca-bin/ocashort?id=2ZET OCA]




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==Contributors==
=='''Contributors'''==
Pernelle Klein, Myriam Deshaies
Pernelle Klein, Myriam Deshaies
Retrieved from "https://proteopedia.org/Sandbox_Reserved_706"