Abstract
Rab5 is a small GTPase that regulates early endosome fusion. We present here the crystal structure of the Rab5 GTPase domain in complex with a GTP analog and the C-terminal domain of effector Rabaptin5. The proteins form a dyad-symmetric Rab5–Rabaptin52–Rab5 ternary complex with a parallel coiled-coil Rabaptin5 homodimer in the middle. Two Rab5 molecules bind independently to the Rabaptin5 dimer using their switch and interswitch regions. The binding does not involve the Rab complementarity-determining regions. We also present the crystal structures of two distinct forms of GDP–Rab5 complexes, both of which are incompatible with Rabaptin5 binding. One has a dislocated and disordered switch I but a virtually intact switch II, whereas the other has its β-sheet and both switch regions reorganized. Biochemical and functional analyses show that the crystallographically observed Rab5–Rabaptin5 complex also exists in solution, and disruption of this complex by mutation abrogates endosome fusion.
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Acknowledgements
The authors thank N. Wakeham and W. Radosevich for technical assistance, the staff of the F1 beamline at CHESS and H. Bellamy at CAMD for help in data collection and T. Mather for critical reading of this manuscript. We are grateful to G. Grenningloh for the Rabaptin5 cDNA. DNA oligomer synthesis and mass spectroscopic analysis were done by the Molecular Biology Resource Facility at University of Oklahoma Health Sciences Center. Confocal images were taken at the Imaging Facility of the Oklahoma Medical Research Foundation. This work was supported in part by a US National Science Foundation career award to G.L.
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Supplementary information
Supplementary Fig. 1
Schematic diagram of the two sets of dyad-symmetric complexes in the Rab5–Rabaptin5(789–862) crystal structure. (PDF 37 kb)
Supplementary Fig. 2
Stereo view of a structural comparison between GTP- and GDP-bound Rab5 structures. (PDF 134 kb)
Supplementary Fig. 3
Chemical cross-link of Rabaptin5. (PDF 479 kb)
Supplementary Fig. 4
Schematic representation of the Rab5–Rabaptin5 interface. (PDF 82 kb)
Supplementary Fig. 5
Stereo view of structural comparison between Rab5 and Rab3A. (PDF 135 kb)
Supplementary Fig. 6
Schematic diagram of the β-sheet re-organization in Arf, Rab5 and Ran upon GTP-hydrolysis. (PDF 63 kb)
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Zhu, G., Zhai, P., Liu, J. et al. Structural basis of Rab5-Rabaptin5 interaction in endocytosis. Nat Struct Mol Biol 11, 975–983 (2004). https://doi.org/10.1038/nsmb832
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DOI: https://doi.org/10.1038/nsmb832
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