title: Comparative thermodynamic analysis of zinc binding to the His/Cys motif in virion infectivity factor.
abstract: HIV-1 virion infectivity factor (Vif) is an accessory protein that induces the proteasomal degradation of the host restriction factor, apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G). Degradation of APOBEC3G requires the interaction of Vif with Cul5, the scaffold for an E3 ubiquitin ligase. A highly conserved region in HIV-1 Vif termed the HCCH motif binds zinc and is critical for recruitment of Cul5 and degradation of APOBEC3G. To gain thermodynamic and mechanistic insight into zinc binding to diverse Vif proteins, we have employed a combination of isothermal titration calorimetry, analytical ultracentrifugation, and Cul5 pull down assays. The proton linkage of zinc binding to HIV-1 Vif was analyzed under different buffer conditions and consistent with the release of two Cys-thiol protons upon zinc binding, supporting earlier EXAFS studies. Zinc binding to Vif proteins from HIV-1, SIVAgm, HIV-2, and SIVMac followed a trend in which the enthalpy of zinc binding became less favorable and the entropy of zinc binding became more favorable. Using AUC, we determined that zinc induced oligomerization of Vif proteins from HIV-1 and SIVAgm but had little or no effect on the oligomeric properties of Vif proteins from HIV-2 and SIVMac. The zinc dependence of Cul5 recruitment by Vif was investigated. All Vif proteins except HIV-2 Vif required zinc to stabilize the interaction with Cul5. The trends in enthalpy-entropy compensation, zinc-induced oligomerization, and Cul5 recruitment are discussed in terms of the apo conformation of the HCCH motif and the role of zinc in stabilizing the structure of Vif.
authors.affiliation: Department of Biochemistry and Molecular Biology Uniformed Services University of the Health Sciences 4301 Jones Bridge Road, Bethesda, Maryland 20814-4799 United States.
authors.forename: Sudipa
authors.initials: S
authors.lastname: Ghimire-Rijal
authors.forename: Ernest L
authors.initials: EL
authors.lastname: Maynard
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D003545:Cysteine / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism*; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D013816:Thermodynamics; D014461:Ultracentrifugation; D015032:Zinc / Q000378:metabolism*; D054320:vif Gene Products, Human Immunodeficiency Virus / Q000737:chemistry / Q000378:metabolism*
chemical_list: D054320:vif Gene Products, Human Immunodeficiency Virus; C514847:vif protein, Human immunodeficiency virus 1; D006639:Histidine; D015032:Zinc; D003545:Cysteine