Structural basis for Rab GTPase activation by VPS9 domain exchange factors

Abstract

RABEX-5 and other exchange factors with VPS9 domains regulate endocytic trafficking through activation of the Rab family GTPases RAB5, RAB21 and RAB22. Here we report the crystal structure of the RABEX-5 catalytic core in complex with nucleotide-free RAB21, a key intermediate in the exchange reaction pathway. The structure reveals how VPS9 domain exchange factors recognize Rab GTPase substrates, accelerate GDP release and stabilize the nucleotide-free conformation. We further identify an autoinhibitory element in a predicted amphipathic helix located near the C terminus of the VPS9 domain. The autoinhibitory element overlaps with the binding site for the multivalent effector RABAPTIN-5 and potently suppresses the exchange activity of RABEX-5. Autoinhibition can be partially reversed by mutation of conserved residues on the nonpolar face of the predicted amphipathic helix or by assembly of the complex with RABAPTIN-5.

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Figure 1: Structure of the RABEX-5 HB-VPS9 tandem in complex with nucleotide-free RAB21.
Figure 2: Conformational changes accompanying formation of the nucleotide-free complex.
Figure 3: Comparison with the nucleotide-free Sec7–Arf GTPase complex.
Figure 4: Identification of an autoinhibitory element in RABEX-5.
Figure 5: Partial reversal of autoinhibition by formation of the complex with RABAPTIN-5.

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Acknowledgements

We thank the National Synchrotron Light Source X25 beamline staff for assistance with X-ray data collection and J. Goldberg (Memorial Sloan-Kettering Cancer Center) for the coordinates of the nucleotide-free ARF1–Gea2 complex. This work was supported by US National Institutes of Health grants GM56324 and DK60564 (to D.G.L.) and by a Charles A. King Postdoctoral Fellowship (to A.D.).

Author information

A.D. was responsible for biochemical and crystallographic experiments, structure determination and refinement of the structural model. D.G.L. assisted with experimental design, synchrotron data collection and structure determination. A.D. and D.G.L. wrote the manuscript.

Correspondence to David G Lambright.

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Supplementary information

Supplementary Fig. 1

Representative electron density for the RABEX-5-RAB21 complex from a simulated annealing omit map. (PDF 260 kb)

Supplementary Fig. 2

Comparison of RABEX-5 alone and in complex with RAB21. (PDF 254 kb)

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Delprato, A., Lambright, D. Structural basis for Rab GTPase activation by VPS9 domain exchange factors. Nat Struct Mol Biol 14, 406–412 (2007). https://doi.org/10.1038/nsmb1232

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