Interrogating APOBEC3s and Vif with NIMR Technoiogy

人类AP0BEC3G(A3G)通过将cDNA胞嘧啶脱氨基转化为尿嘧啶，使HIV基因组发生突变。这种活动可以灭活艾滋病毒。然而，作为一种防御措施，HIV使用一种名为Vif(病毒感染因子)的辅助蛋白质来降解A3G。Vif需要直接的蛋白质-蛋白质相互作用来中和A3G。虽然最近已经获得了A3G脱氨酶结构域的高分辨结构(包括松尾实验室的两个核磁共振结构)，但全长A3G蛋白及其与 事实证明，DNA或VIF更难以捉摸。APOBEC3F(A3F)是另一个有效的HIV-1限制因子。A3F的结构信息要少得多，因为这种蛋白质没有高分辨率的结构。A3G和A3F的结构和生化研究因其不溶性而受到阻碍。我们最近产生了A3G和A3F变种，它们是折叠的，具有催化活性，可以溶解到足以服用 核磁共振波谱。我们建议用核磁共振技术解决全长A3G的结构、A3F的催化结构域及其与Vif(或Vif片段)的络合物。我们还将询问A3G/F和单链DNA之间的相互作用。我们的目标是从结构上揭示(1)A3G和A3F的分子内和分子间结构域相互作用，(2)它们与单链DNA结合的机制，以及A3G和A3F在Vif结合域上的异同(S)。这些研究是一个更大的 跨学科计划项目，将产生关于这一关键宿主-病原体相互作用的前所未有的分子、生化、生物物理和结构信息。

Human AP0BEC3G (A3G ) mutates the HIV genome by deaminafing cDNA cytosines to uracils. This activity can inactivate HIV. However, as a counter-defense, HIV uses an auxiliary protein called Vif (virion infectivity factor) to degrade A3G. A direct protein-protein interaction is required for Vif to neutralize A3G. Although, high-resolution structures of the deaminase domain of A3G have been achieved recently (including two NMR structures from Matsuo laboratory), structures of the full-length A3G protein and its complexes with DNA or Vif have proven more elusive. APOBEC3F (A3F) is another potent HIV-1 restriction factor. Much less structural information is available for A3F as there is no high-resolution structure for this protein. Structural and biochemical studies of A3G and A3F have been hinderd by their insolubility. We have recently generated A3G and A3F variants that are folded, catalytically acitive and soluble enough to take NMR spectra. We propose to solve structures of full-length A3G, the catalytic domain of A3F and their complexes with Vif (or Vif fragments) using NMR. We will also interrogate the interactions between A3G/F and single-stranded DNA. Our objectives are to reveal structurally (1) intra- and inter-molecular domain-domain interaction of A3G and A3F, (2) their mechanisms for binding ssDNA and (S) similarities and differences between A3G and A3F in their Vif-binding domains. These studies are an integral part of a larger interdisciplinary program project that will generate unprecedented molecular, biochemical, biophysical and structural information on this critical host-pathogen interaction.