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To obtain structural information on the complex of mouse Rab27B and Slac-a, a C-terminally truncated form of the GTPase-deficient mutant Rab27B (Q78L) was used. | To obtain structural information on the complex of mouse Rab27B and Slac-a, a C-terminally truncated form of the GTPase-deficient mutant Rab27B (Q78L) was used. | ||
In fact, full size of Rab27B is 218 amino acids long whereas the truncated form of Rab27B contains only the GTPase domain (residues 1 to 201). <ref>PMID:18940604</ref> <ref>http://oca.weizmann.ac.il/oca-bin/ocaids?id=2zet</ref> | In fact, full size of Rab27B is 218 amino acids long whereas the truncated form of Rab27B contains only the GTPase domain (residues 1 to 201). <ref>PMID:18940604</ref> <ref>http://oca.weizmann.ac.il/oca-bin/ocaids?id=2zet</ref> | ||
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| - | Pq est ce qu’on utilize un mutant? Pour simplifier la cristallo ? | ||
'''Detailed structure:''' | '''Detailed structure:''' | ||
Revision as of 17:26, 4 January 2013
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Structure
Structure of Rab27B
General informations:
To obtain structural information on the complex of mouse Rab27B and Slac-a, a C-terminally truncated form of the GTPase-deficient mutant Rab27B (Q78L) was used. In fact, full size of Rab27B is 218 amino acids long whereas the truncated form of Rab27B contains only the GTPase domain (residues 1 to 201). [1] [2]
Detailed structure:
The structure of Rab27B in the complex contains a central six stranded beta sheet (ß1-6) flanked by five alpha helices (α1–5). Like other Ras-like small GTPases, Rab27B binds with GTP and Mg2+ in the conserved nucleotide-binding site (also see figure 2). Rab27B adopt a globular form in the complex. The ß2 and ß3 loop(residues 55-64) and α3-ß5 loop (residues122-126) of Rab27B are both regions involved in Slac2-a binding. [3]
The complex Rab27B/Slac2-a can only be formed when Rab27B is in a GTP form. This can be explain 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 was observed for GDP-bound Rab27B.
Switch 1 and switch 2 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 its γ-phosphate, and this way also allow Slac2-a binding.(see next paragraph) But in the complex Rab27B/GDP switch 1 and switch 2 exist completely apart from the bound GDP, so that there are no inter-actions with Slac2-a and no binding.
Strucure Slac2-a
General informations:
Slac2-a is a fragment from Slp Homology Domain, residues 1-146 from Melanophilin molecule. This protein is also called Exophilin-3. [4] Slac2-a plays an important role in actin-based melanosome transport in mammalian. In fact, the actin-binding domain of Slac2-a/Melanophilin is required for melanosome distribution in melanocytes.[5]
To obtain structural information on the complex of mouse Rab27B/Slac2-a, a truncated form of Slac2-a was used. This fragment of Slac2-a is the minimum region (residue 1 to 146) of Slac2-a that specifically binds to the GTP-bound form of Rab27B.
Detailed structure:
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. [6] SHD1 is folded into a 66 A ° long helix α1, and SHD2 is composed of three helices (α3–α5). 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 (also see fidure 2). The zinc-binding subdomain contains four short ß strands (ß1–ß4) and loops including three short helices (α2, h1, and h2). The two zinc ions (Zn1 and Zn2) are each coordinated by four conserved cysteine residues: Cys64, Cys67, Cys89, and Cys92 for Zn1 binding, and Cys81, Cys84, Cys104, and Cys107 for Zn2 binding. [7]
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