Structural basis for ubiquitin recognition and autoubiquitination by Rabex-5

Abstract

Rabex-5 is an exchange factor for Rab5, a master regulator of endosomal trafficking. Rabex-5 binds monoubiquitin, undergoes covalent ubiquitination and contains an intrinsic ubiquitin ligase activity, all of which require an N-terminal A20 zinc finger followed immediately by a helix. The structure of the N-terminal portion of Rabex-5 bound to ubiquitin at 2.5-Å resolution shows that Rabex-5–ubiquitin interactions occur at two sites. The first site is a new type of ubiquitin-binding domain, an inverted ubiquitin-interacting motif, which binds with 29-μM affinity to the canonical Ile44 hydrophobic patch on ubiquitin. The second is a diaromatic patch on the A20 zinc finger, which binds with 22-μM affinity to a polar region centered on Asp58 of ubiquitin. The A20 zinc-finger diaromatic patch mediates ubiquitin-ligase activity by directly recruiting a ubiquitin-loaded ubiquitin-conjugating enzyme.

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Figure 1: Structure of the Rabex-5 A20 ZnF domain and IUIM.
Figure 2: Ubiquitin recognition by Rabex-5.
Figure 3: Conservation of IUIMs and their relationship to UIMs.
Figure 4: Conservation of A20 ZnF domains.
Figure 5: The ubiquitin–Rabex-5 interaction in vitro.
Figure 6: Ubc recruitment and ubiquitin-ligase activity of Rabex-5 and its mutants.

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Acknowledgements

We thank M. Horning and X. Zhu for outstanding technical assistance, A. Saxena and the staff of the National Synchrotron Light Source, Brookhaven National Laboratory beamline X12C for assistance with data collection and J. Kim for discussions. This research was supported by the intramural program of the NIH through the National Institute of Diabetes and Digestive and Kidney Diseases (J.H.H.), National Cancer Institute (A.M.W.) and National Institute of Child Health and Human Development (J.S.B.). M.S.K. is a PRAT fellow of the National Institute of General Medical Sciences in the NIH. Research carried out at the National Synchrotron Light Source was supported by the US Department of Energy (DOE), Division of Materials Sciences and Division of Chemical Sciences, under Contract No. DE-AC02-98CH10886.

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Correspondence to James H Hurley.

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Supplementary information

Supplementary Fig. 1

Structure of the Rabex-5(9–73):Ubiquitin complex (PDF 747 kb)

Supplementary Fig. 2

A speculative model for ubiquitin-loaded Ubc binding to Rabex-5(9–73) (PDF 240 kb)

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Lee, S., Tsai, Y., Mattera, R. et al. Structural basis for ubiquitin recognition and autoubiquitination by Rabex-5. Nat Struct Mol Biol 13, 264–271 (2006). https://doi.org/10.1038/nsmb1064

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