Conformational analysis of a peptide approximating the HCCH motif in HIV-1 Vif.
Virion infectivity factor (Vif) is an accessory protein encoded by HIV-1. Vif recruits a Cul5-based ubiquitin ligase that targets APOBEC3G, a host-encoded antiviral enzyme, for proteasomal degradation. The C-terminus of Vif contains a conserved His-X(5)-Cys-X(17-18)-Cys-X(3-5)-His (HCCH) motif that binds zinc and interacts with Cul5. In this study, CD spectroscopy, fluorescence spectroscopy, light scattering, and zinc binding assays were used to examine the conformational properties of HCCHp, a 42-amino acid peptide encompassing the HCCH motif. A single tryptophan residue was engineered into HCCHp to probe local structural changes induced by zinc binding. Zinc binding increased burial of the Trp residue from solvent and increased tertiary packing. The solvent 2,2,2-trifluoroethanol (TFE) induced the formation of an alpha-helical conformation of HCCHp with a midpoint of 20% (vol/vol) and inhibited zinc-induced aggregation of HCCHp. TFE titration data were sigmoidal, consistent with the cooperative nature of helix formation. Zinc binding to HCCHp in 30% TFE solutions was cooperative and weakened the TFE-induced structure. In 80% TFE solutions this cooperativity was lost, suggesting a mechanism in which monomeric and oligomeric peptide forms display different affinities for zinc. TFE weakened zinc binding to HCCHp by two orders of magnitude relative to the zinc binding affinity measured in aqueous solvent. The data suggest that HCCHp conformation and zinc binding affinity are tightly coupled. We propose that the lack of intrinsic structure in the HCCH motif may be important for Vif's function as an E3 ubiquitin ligase adaptor protein.
Giri
Kalyan
K
Department of Biochemistry and Molecular Biology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814, USA.
Maynard
Ernest L
EL
D020816:Amino Acid Motifs; D002863:Chromogenic Compounds / Q000737:chemistry; D006801:Humans; D010455:Peptides / Q000737:chemistry / Q000235:genetics; D017433:Protein Structure, Secondary; D012118:Resorcinols / Q000737:chemistry; D014364:Tryptophan / Q000737:chemistry; D015032:Zinc / Q000737:chemistry; D054320:vif Gene Products, Human Immunodeficiency Virus / Q000737:chemistry / Q000235:genetics
D002863:Chromogenic Compounds; D010455:Peptides; D012118:Resorcinols; D054320:vif Gene Products, Human Immunodeficiency Virus; D014364:Tryptophan; D015032:Zinc; C028398:4-(2-pyridylazo)resorcinol