Assembly of HIV-1 Vif-Cul5 E3 ubiquitin ligase through a novel zinc-binding domain-stabilized hydrophobic interface in Vif

Virion infectivity factor (Vif) is an accessory protein encoded by HIV-1 and is critical for viral infection of the host CD4 ؉ T cell population. Vif induces ubiquitination and subsequent degradation of Apo3G, a cytosolic cytidine deaminase that otherwise targets the retroviral genome. Interaction of Vif with the cellular Cullin5-based E3 ubiquitin ligase requires a conserved BC box and upstream residues that are part of the conserved H-(Xaa)5-C-(Xaa)17-18-C-(Xaa)3-5-H (HCCH) motif. The HCCH motif is involved in stabilizing the Vif-Cullin 5 interaction, but the exact role of the conserved His and Cys residues remains elusive. In this report, we find that full-length HIV-1 Vif, as well as a HCCH peptide, is capable of binding to zinc with high specificity. Zinc binding induces a conformational change that leads to the formation of large protein aggregates. EDTA reversed aggregation and regenerated the apoprotein conformation. Cysteine modification studies with the HCCH peptide suggest that C114 is critical for stabilizing the fold of the apopeptide, and that C133 is located in a solvent-exposed region with no definite secondary structure. Selective alkylation of C133 reduced metal-binding specificity of the HCCH peptide, allowing cobalt to bind with rates comparable to that with zinc. This study demonstrates that the HCCH motif of HIV-1 Vif is a unique metal-binding domain capable of mediating protein-protein interactions in the presence of zinc and adds to a growing list of examples in which metal ion binding induces protein misfolding and/or aggregation.aggregation ͉ cullin ubiquitin ligase ͉ metal-binding protein

Section: Resultssupporting

confidence: 93%

“…Several reports (9,10,12,(14)(15)(16)(17)(18)(19) have shown that mutations in the recently defined HCCH motif impair Vif function. In one report (19), Vif C114 (amino acid numbering used hereafter refers to the HIV-1 HXB2 Vif sequence) was proposed to constitute part of a cysteine protease active site involved in gp41 processing.…”

mentioning

confidence: 99%

Zinc binding to the HCCH motif of HIV-1 virion infectivity factor induces a conformational change that mediates protein–protein interactions

Paul

Cui

Maynard

2006

“…Whether a substrate receptor recruits Cul2 or Cul5 depends on an additional Cullin binding interface. In the case of cellular substrate receptors, Cullin selection is determined by the presence of a VHL or SOCS box motif (11), in the case of HIV and SIV, a zinc-stabilized helix mediates Cul5 selection (12)(13)(14)(15)). …”

mentioning

confidence: 99%

Regulation of Hsp90 client proteins by a Cullin5-RING E3 ubiquitin ligase

Ehrlich

Wang

Luo

et al. 2009

Self Cite

109

106

We report a link between Cullin5 (Cul5) E3 ubiquitin ligase and the heat shock protein 90 (Hsp90) chaperone complex. Hsp90 participates in the folding of its client proteins into their functional conformation. Many Hsp90 clients have been reported to be aberrantly expressed in a number of cancers. We demonstrate Cul5 interaction with members of the Hsp90 chaperone complex as well as the Hsp90 client, ErbB2. We observed recruitment of Cul5 to the site of ErbB2 at the plasma membrane and subsequent induction of polyubiquitination and proteasomal degradation. We also demonstrate Cul5 involvement in regulation of another Hsp90 client, Hif-1␣. We observed Cul5 degradation of ErbB2 to occur independently of ElonginB-ElonginC function. The involvement of Cul5 in Hsp90 client regulation has implications in the effectiveness of Hsp90 targeted chemotherapy, which is currently undergoing clinical trials. The link between Cul5 and Hsp90 client regulation may represent an avenue for cancer drug development.chaperone ͉ erbb2

“…The BC box motif creates a hydrophobic interface for binding to EloC. The Cullin box has a specific site for binding to Cul5, which involves an interaction between the highly conserved HCCH zinc-binding motif in Vif and the N-terminal domain (NTD) of Cul5 (22,23).…”

mentioning

confidence: 99%

HIV-1 Vif-mediated ubiquitination/degradation of APOBEC3G involves four critical lysine residues in its C-terminal domain

Iwatani

Chan

Liu

et al. 2009

During coevolution with the host, HIV-1 developed the ability to hijack the cellular ubiquitin/proteasome degradation pathway to counteract the antiviral activity of APOBEC3G (A3G), a host cytidine deaminase that can block HIV-1 replication. Abrogation of A3G function involves the HIV-1 Vif protein, which binds A3G and serves as an adapter molecule to recruit A3G to a Cullin5-based E3 ubiquitin ligase complex. Structure-guided mutagenesis of A3G focused on the 14 most surface-exposed Lys residues allowed us to identify four Lys residues (Lys-297, 301, 303, and 334) that are required for Vif-mediated A3G ubiquitination and degradation.