Structure-based in silico identification of ubiquitin-binding domains provides insights into the ALIX-V:ubiquitin complex and retrovirus budding.
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 μM. We show that ALIX-V oligomerizes with a Hill coefficient of 5.4 and IC(50) of 27.6 μM 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.
Department of Biochemistry and Molecular Biology and Rich Institute for Structural Biology, George S. Wise Faculty of Life Sciences Tel Aviv University, Tel Aviv, Israel.
Tal
T
Keren-Kaplan
Ilan
I
Attali
Michael
M
Estrin
Lillian S
LS
Kuo
Efrat
E
Farkash
Moran
M
Jerabek-Willemsen
Noa
N
Blutraich
Shay
S
Artzi
Aviyah
A
Peri
Eric O
EO
Freed
Haim J
HJ
Wolfson
Gali
G
Prag
D000818:Animals; D006801:Humans; D004860:Infectious Anemia Virus, Equine / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008954:Models, Biological; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D017434:Protein Structure, Tertiary; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D057074:Virus Release / Q000502:physiology*
D009097:Multienzyme Complexes; D044767:Ubiquitin-Protein Ligases