Structure and dynamics of a functional cavity in the HIV-1 Envelope, and its role in conformational changes required for infection

HIV-1包膜功能腔的结构和动力学及其在构象变化中的作用 感染所需的 蛋白质结构中的通道和空腔通常在调节结构、功能和生物学特性中起重要作用。 动力学HIV-1包膜（Env）糖蛋白，一种利用受体结合的构象机器 介导的构象变化以实现病毒和宿主细胞膜融合， 渠道一个高度保守的空腔-称为Phe 43空腔-与受体CD 4的Phe 43残基接合 并且对于CD 4诱导的Env构象转变至关重要。Phe 43空腔也是 广泛和有效的抗体，药物和HIV-1进入肽抑制剂。CD 4模拟微蛋白M48 U1， 广泛有效地中和HIV-1，以pM亲和力结合HIV-1 gp 120，显示出作为阴道给药的功效。 在动物模型中的杀微生物剂，最近，被证明与药物替诺福韦协同抑制艾滋病毒 感染活化的PBMC和人宫颈阴道组织培养物。M48 U1插入甲氧基环己基 Phe 43的分子量为1000。M48 U1及其类似物是唯一一类深入到细胞内的配体。 Phe 43腔，这一特性使它们成为探测深部结构的有用工具，否则就不是 我们之前已经利用M48 U1及其类似物来探测Phe的结构， gp 120单体中的43个空腔。与HIV-1 Env gp 120结合的M48 U1的高分辨率结构显示配体 灵活性，以及适应不同HIV-1分离株的Phe 43空腔的能力， 腔结构的扰动是活性的关键决定因素。 在这项资助中，我们计划利用以下方法探索封闭的HIV-1 Env三聚体中Phe 43空腔的结构： M48 U1分子探针。这项资助的创新来自于我们使用M48 U1及其类似物作为 Phe 43腔的分子探针，来自冷冻EM技术的进步，包括改进的 样品玻璃化方法，改进的显微镜硬件，高通量数据的自动化方法 收集和先进的数据处理算法。这些进步最近使我们能够 建立了一条快速测定HIV-1 Env复合物高分辨率结构的管道。科学 这项资助的前提是，Phe 43空腔是HIV-1 Env中的关键功能组分，是HIV-1 Env的核心。 CD 4受体和许多广泛而有效的抗体和药物的活性。M48 U1是一款 有效的HIV-1进入抑制剂，和唯一的配体之一，填补整个Phe-43腔。探索 当与M48 U1结合时，HIV-1 Env的结构将提供对结构和构象的深入了解。 在Env的闭合状态下对腔体的约束。

Structure and dynamics of a functional cavity in the HIV-1 Envelope, and its role in conformational changes required for infection Channels and cavities in protein structures often play an important role in modulating structure, function and dynamics. The HIV-1 Envelope (Env) glycoprotein, a conformational machine that utilizes receptor binding mediated conformational changes to effect virus and host cell membrane fusion, is riddled with cavities and channels. A highly conserved cavity – called the Phe43 cavity - engages the Phe43 residue of receptor CD4 and is critical for CD4-induced Env conformational transitions. The Phe43 cavity is also the binding site of broad and potent antibodies, drugs and peptide inhibitors of HIV-1 entry. The CD4-mimetic miniprotein M48U1, effects broad and potent HIV-1 neutralization, binds HIV-1 gp120 with pM affinity, showed efficacy as a vaginal microbicide in animal models, and more recently, was shown to synergize with the drug Tenofovir to inhibit HIV infection in activated PBMCs and human cervicovaginal histocultures. M48U1 inserts a methoxy cyclohexyl moiety to fill the Phe43 cavity. M48U1 and its analogs are the only class of ligands that reach deep into the Phe43 cavity, a property that makes them valuable tools for probing the structure of a deep, otherwise not easily accessible cavity.We have previously utilized M48U1 and its analogs to probe the structure of the Phe- 43 cavity in gp120 monomers. High resolution structures of M48U1 bound to HIV-1 Env gp120 revealed ligand flexibility, and the ability to fit within and adapt to the Phe43 cavities of diverse HIV-1 isolates with minimal perturbations of the cavity structure are key determinants of activity. In this grant we propose to explore the structure of the Phe43 cavity in the closed HIV-1 Env trimer using M48U1 as a molecular probe. The innovation in this grant derives from our use of M48U1 and its analogs as molecular probes of the Phe43 cavity, from the advances in cryo-EM technology that include improved specimen vitrification methods, improved microscope hardware, automated methods for high-throughput data collection, and advanced algorithms for data processing. These advances have recently allowed us to establish a rapid pipeline for determining high resolution structures of HIV-1 Env complexes. The scientific premise of this grant is that the Phe43 cavity is a critical functional component in HIV-1 Env, central to the activity of the CD4 receptor and many broad and potent antibodies and drugs. M48U1 is an exceptionally effective HIV-1 entry inhibitor, and one of the only ligands that fills the entire Phe-43 cavity. Exploring the structures of HIV-1 Env when bound to M48U1 will provide insights into the structure and the conformational constraints on the cavity in the closed state of the Env.