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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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.).
The authors declare no competing financial interests.
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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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