Comparative Thermodynamic Analysis of Zinc Binding to the His/Cys Motif in Virion Infectivity Factor
Comparative Thermodynamic Analysis of Zinc Binding to the His/Cys Motif in Virion Infectivity Factor
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DOI：
10.1021/ic402907g
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发表时间：
2014-05-05
影响因子：
4.6
通讯作者：
Maynard, Ernest L., Jr.
中科院分区：
文献类型：
--
作者：
Ghimire-Rijal, Sudipa;Maynard, Ernest L., Jr.
HIV-1病毒粒子感染因子（Vif）是一种辅助蛋白，可诱导宿主限制性因子--载脂蛋白B mRNA编辑酶催化多肽样3G（APOBEC 3G）的蛋白酶体降解。APOBEC 3G的降解需要Vif与Cul 5的相互作用，Cul 5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，与锌结合，对Cul 5的募集和APOBEC 3G的降解至关重要。为了获得锌与不同Vif蛋白结合的热力学（1）和机制，我们采用了等温滴定量热法，分析超离心和Cul 5下拉测定的组合。在不同的缓冲液条件下分析了锌与HIV-1 Vif结合的质子连接，这与锌结合后释放两个Cys-巯基质子一致，支持早期的EXAFS研究。锌与来自HIV-1、SIVAgm、HIV-2和SIVMac的Vif蛋白的结合遵循锌结合的焓变得不太有利而锌结合的熵变得更有利的趋势。使用AUC，我们确定锌诱导HIV-1和SIVAgm的Vif蛋白的寡聚化，但对HIV-2和SIVmac的Vif蛋白的寡聚性质几乎没有影响。研究了Vif募集Cul 5的锌依赖性。除HIV-2 Vif外，所有Vif蛋白都需要锌来稳定与Cul 5的相互作用。的趋势，熵补偿，锌诱导的寡聚化，和Cul 5招聘的HCCH基序的载脂蛋白构象和锌的作用，在稳定Vif的结构方面进行了讨论。
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(1) 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.