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TBC-domain GAPs for Rab GTPases accelerate GTP hydrolysis by a dual-finger mechanism

Abstract

Rab GTPases regulate membrane trafficking by cycling between inactive (GDP-bound) and active (GTP-bound) conformations1. The duration of the active state is limited by GTPase-activating proteins (GAPs), which accelerate the slow intrinsic rate of GTP hydrolysis. Proteins containing TBC (Tre-2, Bub2 and Cdc16) domains are broadly conserved in eukaryotic organisms and function as GAPs for Rab GTPases as well as GTPases that control cytokinesis2. An exposed arginine residue is a critical determinant of GAP activity in vitro and in vivo3,4,5. It has been expected that the catalytic mechanism of TBC domains would parallel that of Ras and Rho family GAPs. Here we report crystallographic, mutational and functional analyses of complexes between Rab GTPases and the TBC domain of Gyp1p. In the crystal structure of a TBC-domain–Rab-GTPase–aluminium fluoride complex, which approximates the transition-state intermediate for GTP hydrolysis, the TBC domain supplies two catalytic residues in trans, an arginine finger analogous to Ras/Rho family GAPs and a glutamine finger that substitutes for the glutamine in the DxxGQ motif of the GTPase. The glutamine from the Rab GTPase does not stabilize the transition state as expected but instead interacts with the TBC domain. Strong conservation of both catalytic fingers indicates that most TBC-domain GAPs may accelerate GTP hydrolysis by a similar dual-finger mechanism.

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Figure 1: Identification of mammalian Rab GTPase substrates for the Gyp1p TBC domain.
Figure 2: Structure of the Gyp1p TBC domain in complex with Rab33–GDP–AlF3.
Figure 3: Network of polar interactions in the AlF 3 -binding site and comparison with other GAP–GTPase complexes.
Figure 4: Mutational analysis of the Gyp1p Rab interaction epitope.

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Acknowledgements

We thank P. Novick for the GYP1-null strains and plasmids, and J. Saporita for assistance with yeast experiments. This work was supported by a National Institutes of Health grant.

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Correspondence to David G. Lambright.

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Competing interests

Coordinates and structure factors for the Gyp1p–Rab33–AlF3 complex have been deposited with the Protein Data Bank under the ID code 2G77. Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.

Supplementary information

Supplementary Figure S1

Identification of Rab GTPase substrates for the Gyp1p TBC domain. (PDF 113 kb)

Supplementary Figure S2

Gyp1p and Rab33 form a complex in the presence of aluminum fluoride. (PDF 190 kb)

Supplementary Figure S3

Additional information relevant to structure determination. (PDF 347 kb)

Supplementary Figure S4

Conservation and variability within the interface between Gyp1p and Rab33. (PDF 307 kb)

Supplementary Figure S5

Comparison of the structures of Gyp1p and Rab33 alone and in the complex. (PDF 216 kb)

Supplementary Figure S6

Kinetics of GTP hydrolysis catalyzed by the Gyp1p and Gyp7p TBC domains. (PDF 286 kb)

Supplementary Figure S7

Western blot analysis of wild type and mutant Gyp1p expression. (PDF 145 kb)

Supplementary Table S1

Rab constructs. (PDF 37 kb)

Supplementary Table S2

Data collection, phasing, and refinement statistics. (PDF 77 kb)

Supplementary Methods

This file contains supplementary methods, including one table and eight references. (PDF 106 kb)

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Pan, X., Eathiraj, S., Munson, M. et al. TBC-domain GAPs for Rab GTPases accelerate GTP hydrolysis by a dual-finger mechanism. Nature 442, 303–306 (2006). https://doi.org/10.1038/nature04847

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