Molecular Structure and Biochemical Properties of the HCCH−Zn2+Site in HIV-1 Vif

Kalyan Giri, Robert A. Scott, Ernest L. Maynard
2009 Biochemistry  
Virion infectivity factor (Vif) is an HIV accessory protein that is essential for the infection of CD4 + T cells. Vif recruits a Cullin-5 (Cul5)-based ubiquitin ligase that targets a host cytidine deaminase, apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3G (APOBEC3G), for proteasomal degradation. The Vif N-terminus binds APOBEC3G and the C-terminus interacts with the Cul5-based ubiquitin ligase machinery. Within the C-terminus, a highly conserved H 108 -X 5 -C 114 -X 17-18 -C
more » ... 33 -X 3-5 -H 139 (HCCH) motif binds zinc and is implicated in the Vif-Cul5 interaction. We have employed the biomimetic peptide HCCHp (HIV-1 Vif amino acids 101-142) in order to determine the zinc ligands and investigate the role of zinc binding in Cul5 recognition. Using CD spectroscopy, a competitive zinc binding assay, and a light scattering assay, we found that mutation of the conserved His and Cys residues in HCCHp had little effect on secondary structure, but reduced zinc-binding affinity and altered the aggregation properties of the peptides. X-ray absorption spectroscopy was used to study zinc coordination in wild type HCCHp. The data are consistent with S 2 N(imid) 2 coordination and strongly suggest that His-108, Cys-114, Cys-133, and His-139 are zinc ligands. Mutation of one or both conserved Cys residues in HCCHp led to a decrease in Cys ligation, and an increase in the number of (N,O) ligands, with noninteger coordination numbers suggesting zinc site heterogeneity. A purified fragment of human Cul5 was found to inhibit zinc-induced aggregation of HCCHp, and pull-down experiments revealed that zinc binding to HCCHp increases the strength of the HCCHp-Cul5 interaction by eight-fold. HIV-1 accessory proteins modulate the host cellular environment to permit viral replication and transmission (reviewed in (1-4) ). The HIV-1 vif gene encodes virion infectivity factor (Vif), 1 a 23-kDa basic protein of 192 amino acids. Vif is expressed late in the viral life cycle † This work was supported by an intramural grant to E.L.M. and an NIH grant (GM42025) to R.A.S. Portions of this research were carried out at the Stanford Synchrotron Radiation Lightsource, a national user facility operated by Stanford University on behalf of the U.S. . SUPPORTING INFORMATION CD spectroscopic analysis of human Cul5(1-384) and detailed XAS/EXAFS curve-fitting results for all HCCHp samples. This material is available free of charge via the Internet at http://pubs.acs.org.
doi:10.1021/bi900677w pmid:19588889 pmcid:PMC2729716 fatcat:pbzm3sdtjbhitbqtkc3nuc4xiu