期刊
EMBO JOURNAL
卷 32, 期 4, 页码 538-551出版社
WILEY
DOI: 10.1038/emboj.2013.4
关键词
computational analyses; in silico scan; protein trafficking; ubiquitin; virus budding
资金
- Israeli Science Foundation [1695/08, 464/11]
- EC FP7 Marie Curie International Reintegration Grant [PIRG03-GA-2008-231079]
- Israeli Ministry of Health [5108]
- Marianna and Jorge Saia Fund for HIV and Parkinson Diseases
- Center for Cancer Research, National Cancer Institute, NIH
- Intramural AIDS Targeted Antiviral Programme
- Israel Science Foundation (ISF) [1403/09]
- Minerva-Minkowski Center for Geometry
- Adams Fellowship Fund
The ubiquitylation signal promotes trafficking of endogenous and retroviral transmembrane proteins. The signal is decoded by a large set of ubiquitin (Ub) receptors that tether Ub-binding domains (UBDs) to the trafficking machinery. We developed a structure-based procedure to scan the protein data bank for hidden UBDs. The screen retrieved many of the known UBDs. Intriguingly, new potential UBDs were identified, including the ALIX-V domain. Pull-down, cross-linking and E3-independent ubiquitylation assays biochemically corroborated the in silico findings. Guided by the output model, we designed mutations at the postulated ALIX-V: Ub interface. Biophysical affinity measurements using microscale-thermophoresis of wild-type and mutant proteins revealed some of the interacting residues of the complex. ALIX-V binds mono-Ub with a K-d of 119 mu M. We show that ALIX-V oligomerizes with a Hill coefficient of 5.4 and IC50 of 27.6 mu M and that mono-Ub induces ALIX-V oligomerization. Moreover, we show that ALIX-V preferentially binds K63 di-Ub compared with mono-Ub and K48 di-Ub. Finally, an in vivo functionality assay demonstrates the significance of ALIX-V: Ub interaction in equine infectious anaemia virus budding. These results not only validate the new procedure, but also demonstrate that ALIX-V directly interacts with Ub in vivo and that this interaction can influence retroviral budding. The EMBO Journal (2013) 32, 538-551. doi: 10.1038/emboj.2013.4; Published online 29 January 2013
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