Ghimire-Rijal Sudipa, Maynard Ernest L
Department of Biochemistry and Molecular Biology Uniformed Services University of the Health Sciences 4301 Jones Bridge Road, Bethesda, Maryland 20814-4799 United States.
Inorg Chem. 2014 May 5;53(9):4295-302. doi: 10.1021/ic402907g. Epub 2014 Apr 16.
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.
HIV-1病毒体感染性因子（Vif）是一种辅助蛋白，可诱导宿主限制因子载脂蛋白B信使核糖核酸编辑酶催化多肽样3G（APOBEC3G）的蛋白酶体降解。APOBEC3G的降解需要Vif与Cul5相互作用，Cul5是E3泛素连接酶的支架。HIV-1 Vif中一个高度保守的区域称为HCCH基序，可结合锌，对Cul5的募集和APOBEC3G的降解至关重要。为了深入了解锌与多种Vif蛋白结合的热力学和机制，我们采用了等温滴定量热法、分析超速离心法和Cul5下拉试验相结合的方法。在不同缓冲条件下分析了锌与HIV-1 Vif结合的质子连接情况，结果与锌结合时两个半胱氨酸硫醇质子的释放一致，支持了早期的扩展X射线吸收精细结构研究。锌与来自HIV-1、猴免疫缺陷病毒（SIVAgm）、HIV-2和猴免疫缺陷病毒（SIVMac）的Vif蛋白结合呈现出一种趋势，即锌结合的焓变变得不那么有利，而锌结合的熵变变得更有利。使用分析超速离心法，我们确定锌诱导HIV-1和SIVAgm的Vif蛋白发生寡聚，但对HIV-2和SIVMac的Vif蛋白的寡聚特性几乎没有影响。研究了Vif对Cul5募集的锌依赖性。除HIV-2 Vif外，所有Vif蛋白都需要锌来稳定与Cul5的相互作用。根据HCCH基序的脱辅基构象以及锌在稳定Vif结构中的作用，讨论了焓-熵补偿、锌诱导的寡聚和Cul5募集的趋势。
