Molecular structure and biochemical properties of the HCCH-Zn2+ site in HIV-1 Vif.
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(133)-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 8-fold.
Giri
Kalyan
K
Department of Biochemistry and Molecular Biology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814, USA.
Scott
Robert A
RA
Maynard
Ernest L
EL
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D032701:Biomimetics / Q000379:methods; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D017124:Conserved Sequence; D044842:Cullin Proteins / Q000302:isolation & purification / Q000378:metabolism; D016346:Gene Products, vif / Q000737:chemistry* / Q000378:metabolism; D015497:HIV-1 / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping; D015032:Zinc / Q000737:chemistry* / Q000378:metabolism
C496871:CUL5 protein, human; D002352:Carrier Proteins; D044842:Cullin Proteins; D016346:Gene Products, vif; D010455:Peptides; C017800:zinc-binding protein; D015032:Zinc