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Crystal structure of the small GTPase Rab27B complexed with the Slp homology domain of Melanophilin

PDB ID 2zet

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2zet, resolution 3.00Å ()
Ligands: , , ,
Non-Standard Residues:
Gene: Rab27b (Mus musculus), Slac2a (Mus musculus)
Resources: FirstGlance, OCA, RCSB, PDBsum, TOPSAN
Coordinates: save as pdb, mmCIF, xml




Contents

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 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.

Figure1: Cycle of the small G protein Rab
Figure1: Cycle of the small G protein Rab [1]

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.[2]

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.[3]

Biological functions

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 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.[4]

Exophilin3/Slac2-a/Melanophilin

As written before, melanophilin is a Rab27A/B effector. Slac2-a acts in the regulation of melanosome transport in mammalian skin melanocytes by binding melanosome-bound Rab27A and myosin Va, an actin-based motor protein. Although Slac2-a directly interacts with Rab27A and myosin Va via its N-terminal region (amino acids 1 to 146) and the middle region (amino acids 241 to 405), respectively, the functional importance of the putative actin-binding domain of the Slac2-a C terminus (amino acids 401 to 590) in melanosome transport has never been elucidated.[5]

Rab27B complexed with the Slp homology domain of Slac2-a

Little is known about the interactions between Rab27B and melanophilin. The Rab27B/Slac2-a complex has several intermolecular hydrogen bonds and electrostatic interactions. It may be used for drugs development for the Griscelli syndrome.[6]


Structures

2ZET is a 4 chains structure of sequences from Mus musculus. Full crystallographic information is available from OCA.

Structure of Rab27B

Figure 2: Overall stucture of the Rab27B/Slac2-a complex.
Figure 2: Overall stucture of the Rab27B/Slac2-a complex. [7]

General informations:

To obtain structural informations on the complex of mouse Rab27B and Slac2-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). [8] [9]


Detailed structure:

The structure of Rab27B in the complex contains a (ß1-6) flanked by (α1–5). Like other Ras-like small GTPases, Rab27B binds with and in the conserved nucleotide-binding site (also see figure 2). Rab27B adopts a globular form in the complex. The and of Rab27B are both regions involved in Slac2-a binding. [10]

Figure 3: Structural comparaison of Rab27B in GTP and GDP form.
Figure 3: Structural comparaison of Rab27B in GTP and GDP form. [11]

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. and 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

General informations:

Slac2-a is a fragment from Slp Homology Domain, residues 1-146 from Melanophilin molecule. This protein is also called Exophilin-3. [12] 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.[13]

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. [14] SHD1 is folded into a 66 Å , and SHD2 is composed of . 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 contains four short  Beta Strands  (ß1–ß4) and five loops(L1-L5), including one short Alpha Helices α2 and three short helices (in purple). The are each coordinated by four conserved cysteine residues: for Zn1 binding, and for Zn2 binding. [15]

Final model of 2zet: Two Rab27B/Slac2-a complexes (A/C and B/D)

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Structure of the complex

Model and general structure :

The final model of 2ZET includes:

- two pairs of the Rab27B/Slac2-a complex ,called and complex (A and B designate Rab 27B and C and D designate Slac2-a),

- ,

- ,

- ,

- .

Because the structures of the two Rab27B/Slac2-a complex are the same, we will describe only the structure of A/C complex with for ligand one GTP molecule, one magnesium ion, two zinc ions and one sulfate ion.

The A/C complex is a 3.0 Å crystal structure of the effector domain of mouse Slac2-a complexed with a GTPase-deficient mutant (Q78L) of mouse Rab27B. Rab27B adopts a globular form in the complex and Slac2-a forms an elongated helical fold with a subdomain that contains two zinc ions.


Interface of Rab27B/Slac2-a and interaction details:

Rab27B interacts with Slac2-a with a buried surface area of 2534 Ų, which corresponds to 30.2% of the total surface area.

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.

Figure 4: Rab27B/Slac2-a contact interfaces : (A) Contact area 1, (C) Contact area 2, (E) Contact area 3.
Figure 4: Rab27B/Slac2-a contact interfaces : (A) Contact area 1, (C) Contact area 2, (E) Contact area 3.[16]
  • The first contact area of the Rab27B/Slac2-a interface consists of , part of the switch and interswitch regions of Rab27B, and the coiled-coil regions of Slac2-a.[17] In this first contact area we can find three different types of interactions, electrostatic, hydrophobic and hydrogen bonds: (see figure 4A)

- from of Rab27B participate in Hydrophobic interactions with the residues from of Slac2-a. Furthermore, is located in the middle of this contact area, and it plays a central role in Hydrophobic interactions but also for Slac2-a binding. .[18]

-There are also electrostatic interactions involved in this Slac2-a binding, for instance amino acids of Slac2-a are required for this interaction. .[19]

-In the first contact area, hydrogen bonds between Rab27B/Slac2-a were also observed. First, of Slac2-a. Secondly, . Third, carbonyl group from the α4–α5 loop of Slac2-a. To finish, . [20]

  • The second contact area is formed by the Rab complementarity-determining regions helical region (also see figure 4C). [21] [22]

At this interface, there are hydrophobic interactions and three hydrogen bonds. carbonyl group of Rab27B. .

  • The third contact area is the smallest one . It involves the of Slac2-a and the of Rab27B, with superposition of their molecular surfaces (see figure 4E).[23]


These findings strongly suggested that from switch 2 of Rab27 are the minimum determinants of Slac2-a binding.[24]

External Resources



References

  1. ↑ http://www.ncbi.nlm.nih.gov/pmc/articles/PMC138937/
  2. ↑ Kukimoto-Niino M, Sakamoto A, Kanno E, Hanawa-Suetsugu K, Terada T, Shirouzu M, Fukuda M, Yokoyama S. Structural basis for the exclusive specificity of Slac2-a/melanophilin for the Rab27 GTPases. Structure. 2008 Oct 8;16(10):1478-90. PMID:18940604 doi:10.1016/j.str.2008.07.014
  3. ↑ http://www.spring8.or.jp/pdf/en/res_fro/08/028-029.pdf
  4. ↑ http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2043558/
  5. ↑ http://www.ncbi.nlm.nih.gov/pmc/articles/PMC165717/
  6. ↑ http://www.spring8.or.jp/pdf/en/res_fro/08/028-029.pdf
  7. ↑ Kukimoto-Niino M, Sakamoto A, Kanno E, Hanawa-Suetsugu K, Terada T, Shirouzu M, Fukuda M, Yokoyama S. Structural basis for the exclusive specificity of Slac2-a/melanophilin for the Rab27 GTPases. Structure. 2008 Oct 8;16(10):1478-90. PMID:18940604 doi:10.1016/j.str.2008.07.014
  8. ↑ Kukimoto-Niino M, Sakamoto A, Kanno E, Hanawa-Suetsugu K, Terada T, Shirouzu M, Fukuda M, Yokoyama S. Structural basis for the exclusive specificity of Slac2-a/melanophilin for the Rab27 GTPases. Structure. 2008 Oct 8;16(10):1478-90. PMID:18940604 doi:10.1016/j.str.2008.07.014
  9. ↑ http://oca.weizmann.ac.il/oca-bin/ocaids?id=2zet
  10. ↑ Kukimoto-Niino M, Sakamoto A, Kanno E, Hanawa-Suetsugu K, Terada T, Shirouzu M, Fukuda M, Yokoyama S. Structural basis for the exclusive specificity of Slac2-a/melanophilin for the Rab27 GTPases. Structure. 2008 Oct 8;16(10):1478-90. PMID:18940604 doi:10.1016/j.str.2008.07.014
  11. ↑ Kukimoto-Niino M, Sakamoto A, Kanno E, Hanawa-Suetsugu K, Terada T, Shirouzu M, Fukuda M, Yokoyama S. Structural basis for the exclusive specificity of Slac2-a/melanophilin for the Rab27 GTPases. Structure. 2008 Oct 8;16(10):1478-90. PMID:18940604 doi:10.1016/j.str.2008.07.014
  12. ↑ http://oca.weizmann.ac.il/oca-bin/ocaids?id=2zet
  13. ↑ Kuroda TS, Ariga H, Fukuda M. The actin-binding domain of Slac2-a/melanophilin is required for melanosome distribution in melanocytes. Mol Cell Biol. 2003 Aug;23(15):5245-55. PMID:12861011
  14. ↑ Fukuda M, Kuroda TS, Mikoshiba K. Slac2-a/melanophilin, the missing link between Rab27 and myosin Va: implications of a tripartite protein complex for melanosome transport. J Biol Chem. 2002 Apr 5;277(14):12432-6. Epub 2002 Feb 20. PMID:11856727 doi:10.1074/jbc.C200005200
  15. ↑ Kukimoto-Niino M, Sakamoto A, Kanno E, Hanawa-Suetsugu K, Terada T, Shirouzu M, Fukuda M, Yokoyama S. Structural basis for the exclusive specificity of Slac2-a/melanophilin for the Rab27 GTPases. Structure. 2008 Oct 8;16(10):1478-90. PMID:18940604 doi:10.1016/j.str.2008.07.014
  16. ↑ Kukimoto-Niino M, Sakamoto A, Kanno E, Hanawa-Suetsugu K, Terada T, Shirouzu M, Fukuda M, Yokoyama S. Structural basis for the exclusive specificity of Slac2-a/melanophilin for the Rab27 GTPases. Structure. 2008 Oct 8;16(10):1478-90. PMID:18940604 doi:10.1016/j.str.2008.07.014
  17. ↑ Kukimoto-Niino M, Sakamoto A, Kanno E, Hanawa-Suetsugu K, Terada T, Shirouzu M, Fukuda M, Yokoyama S. Structural basis for the exclusive specificity of Slac2-a/melanophilin for the Rab27 GTPases. Structure. 2008 Oct 8;16(10):1478-90. PMID:18940604 doi:10.1016/j.str.2008.07.014
  18. ↑ Kukimoto-Niino M, Sakamoto A, Kanno E, Hanawa-Suetsugu K, Terada T, Shirouzu M, Fukuda M, Yokoyama S. Structural basis for the exclusive specificity of Slac2-a/melanophilin for the Rab27 GTPases. Structure. 2008 Oct 8;16(10):1478-90. PMID:18940604 doi:10.1016/j.str.2008.07.014
  19. ↑ Kukimoto-Niino M, Sakamoto A, Kanno E, Hanawa-Suetsugu K, Terada T, Shirouzu M, Fukuda M, Yokoyama S. Structural basis for the exclusive specificity of Slac2-a/melanophilin for the Rab27 GTPases. Structure. 2008 Oct 8;16(10):1478-90. PMID:18940604 doi:10.1016/j.str.2008.07.014
  20. ↑ Kukimoto-Niino M, Sakamoto A, Kanno E, Hanawa-Suetsugu K, Terada T, Shirouzu M, Fukuda M, Yokoyama S. Structural basis for the exclusive specificity of Slac2-a/melanophilin for the Rab27 GTPases. Structure. 2008 Oct 8;16(10):1478-90. PMID:18940604 doi:10.1016/j.str.2008.07.014
  21. ↑ Ostermeier C, Brunger AT. Structural basis of Rab effector specificity: crystal structure of the small G protein Rab3A complexed with the effector domain of rabphilin-3A. Cell. 1999 Feb 5;96(3):363-74. PMID:10025402
  22. ↑ Kukimoto-Niino M, Sakamoto A, Kanno E, Hanawa-Suetsugu K, Terada T, Shirouzu M, Fukuda M, Yokoyama S. Structural basis for the exclusive specificity of Slac2-a/melanophilin for the Rab27 GTPases. Structure. 2008 Oct 8;16(10):1478-90. PMID:18940604 doi:10.1016/j.str.2008.07.014
  23. ↑ Kukimoto-Niino M, Sakamoto A, Kanno E, Hanawa-Suetsugu K, Terada T, Shirouzu M, Fukuda M, Yokoyama S. Structural basis for the exclusive specificity of Slac2-a/melanophilin for the Rab27 GTPases. Structure. 2008 Oct 8;16(10):1478-90. PMID:18940604 doi:10.1016/j.str.2008.07.014
  24. ↑ Kukimoto-Niino M, Sakamoto A, Kanno E, Hanawa-Suetsugu K, Terada T, Shirouzu M, Fukuda M, Yokoyama S. Structural basis for the exclusive specificity of Slac2-a/melanophilin for the Rab27 GTPases. Structure. 2008 Oct 8;16(10):1478-90. PMID:18940604 doi:10.1016/j.str.2008.07.014


Contributors

Pernelle Klein, Myriam Deshaies

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