NMR Structural And Dynamics Studies Of Hiv-1 Protease

HIV-1 蛋白酶的 NMR 结构和动力学研究

• 批准号: 7146110
• 负责人: DENNIS A TORCHIA
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7146110
• 关键词: antiAIDS agent; aspartic endopeptidases; computer simulation; conformation; enzyme activity; enzyme inhibitors; enzyme structure; human immunodeficiency virus 1; mathematical model; nuclear magnetic resonance spectroscopy; protease inhibitor; protein folding; protein structure function

The mature, active HIV-1 protease is a homodimer that is made up of monomer subunits containing 99 amino acid residues. At least eight FDA-approved anti-protease drugs have been developed. Each of these drugs binds tightly to the dimer active site, thereby inhibiting catalytic activity and preventing propagation of the AIDs virus; however, one unfortunate consequence of targeting a single site on the protease has been the emergence of viral strains which carry multi-drug resistant mutations within their protease molecules. For this reason, there is considerable interest in identifying alternative protease sites that are suitable drug targets. Of particular interest are compounds that bind to the interface of the dimer. Such compounds will block formation of the active protease dimer, but be insensitive to current multidrug-resistant protease variants. In spite of these attractive features, dimerizatioin inhibitors face the formidable challenge of dissociating the tightly bound mature protease homodimer. We have shown that, in contrast with the mature protease, protease constructs, among them those that contain N-terminal extensions (similar to those of the protease precursor, which is embedded within the Gag-Pol polyprotein), have a million-fold larger dissociation constants and are predominantly monomeric at concentrations required for NMR studies. These discoveries suggest that the precursor monomer rather than the mature protease monomer is the target of choice of dimerization inhibitors. We have therefore initiated NMR studies to screen for molecules that interact with models of protease precursor monomer constructs, in order to identify inhibitors of precursor dimerization. Previous structural work that we have done on the protease monomer will aid in the design of dimerization inhibitors. The structual studies have shown that the monomer contains a folded core domain that is common to all monomer constructs that we have examined. The core domain presents preorganized target sites to which small compounds can bind. In addition we have found that mutations in core amino acid residues, which are not in the dimer interface, can destabilize the dimer. These sites are therefore also potential targets for dimer inhibitors. In recent work, we have used a combination of fluorescence and high resolution NMR measurements to follow the urea denaturation profiles of various protease mutants that are predominantly monomeric in solution. Preliminary analysis of this data indicates that either a deletion or an extension of several residues at the N- or C- termini of the mature protease sequence, does not affect the observed denaturation profile of the monomer. This result is consistent with our previous structural studies which show that the N- and C- terminal regions of the mature protease monomer and the monomer precursor (whose N-terminus extends beyond that of the mature protease) are disordered and flexible in solution. In contrast with these results, a Ser-Ala substitution near the active site increased the stability of the monomer against urea denaturation. This observation is thought to result from an enhancement of hydrophobic packing involving the Ala methyl with nearby Leu sidechains. NMR measurements of monomer structural features are being used together with urea denaturation profiles to elucidate structure-stability relationships of various mutants. We have continued studies of the dynamics and interactions of the N- and C-terminal strands that form the primary interface of the protease homodimer. This work is aimed at further understanding the mechanism of protease precursor processing. In the static structural model of the protease, derived from X-ray and NNMR work, terminal residues 1-4 and 96-99 from both monomers form a 4-stranded beta-sheet (the primary dimer interface) to which the exposed autolysis susceptible loop, containing residues 5-9, is connected. However, NMR transverse spin relaxation (R2) dispersion data, hydrogen-deuterium exchange rates and two-dimensional lineshapes provide strong evidence that residues 1-9 of the dimer interface sample two conformations, in dynamic equilibrium. The labile nature of the N-terminus, suggested by the data, is consistent with conclusions described in previous reports on a variety of protease mutants, which revealed that interactions involving the solvent exposed N-terminal strands of the protease are much less important in stabilizing the homodimer than interactions involving the interior C-terminal strands. Furthermore, the proposed dynamic structure rationalizes kinetics data which show that the N-terminal strand folds into the active site of the protease precursor. This conformational switch permits intermolecular cleavage of the immature N-terminus (an early step in Gag-Pol processing). Finally, the flexible N-terminus reveals how the loop containing residues 5-9 becomes fully accessible for autoproteolysis. Analysis of R2 relaxation dispersion profiles can, in principle, provide quantitative information about the chemical shifts and populations of conformations in dynamic equilibrium as well as the rate of the conformational interconversion. Accurate values of these physical parameters provide a basis for characterizing the structural and energetic changes associated with the dynamic process. Recently we have shown that extracting the best values of these parameters and their uncertainties requires careful assessment of experimental errors, both random and systematic, as well as correct application of statistical criteria to ensure that the data are fit with the appropriate statistical model. Current research is focusing upon analysis of errors in the dynamic parameters that result from the common assumption that the sites undergoing exchange have identical intrinsic relaxation rates. We have carried out extensive computer simulations that provide accurate quantities values of such errors for a variety of specific cases. In addition, we used the Carver-Richards equation to obtain general theoretical expressions for the errors. The theoretical equations predict results that are in good agreement with those obtained from the numerical calculations, and provide the spectoscopist with simple expressions for estimating the size of the errors under a wide range of experimental conditions.

成熟的活性HIV-1蛋白酶是一种由包含99个氨基酸残基的单体亚基组成的同型二聚体。已经开发了至少八种FDA批准的抗癌药。这些药物中的每一种都与二聚体活性位点紧密结合，从而抑制催化活性并防止艾滋病病毒传播。但是，靶向蛋白酶的单个位点的不幸后果是病毒菌株的出现，这些病毒菌株在其蛋白酶分子中携带多药物抗性突变。因此，人们对识别合适的药物靶标的替代蛋白酶位点具有很大的兴趣。特别感兴趣的是结合二聚体界面的化合物。这样的化合物将阻止活性蛋白酶二聚体的形成，但对当前耐多药的蛋白酶变体不敏感。尽管具有这些吸引人的特征，但二聚肌抑制剂仍面临分离紧密结合的成熟蛋白酶同二聚体的巨大挑战。我们已经表明，与成熟的蛋白酶，蛋白酶构建体相比，其中包含N末端扩展的蛋白酶构建体（类似于蛋白酶前体的蛋白酶前体的蛋白酶构建体，该蛋白酶前体嵌入了GAG-POL多蛋白中的蛋白酶前体）具有较大的分离常数，并且主要是NMR浓度的单体元素。这些发现表明，前体单体而不是成熟的蛋白酶单体是选择二聚化抑制剂的靶标。因此，我们已经启动了NMR研究，以筛选与蛋白酶前体单体构建体相互作用的分子，以鉴定前体二聚化的抑制剂。我们在蛋白酶单体上所做的以前的结构工作将有助于设计二聚化抑制剂。结构研究表明，单体包含一个折叠的核心结构域，这是我们检查的所有单体构建体共有的。核心结构域提出了小型化合物可以结合的预组织目标位点。此外，我们发现不在二聚体界面中的核心氨基酸残基中的突变会破坏二聚体的稳定。因此，这些位点也是二聚体抑制剂的潜在靶标。 在最近的工作中，我们使用了荧光和高分辨率NMR测量值的组合，以遵循各种溶液中主要单体蛋白酶突变体的尿素变性曲线。对该数据的初步分析表明，成熟蛋白酶序列的N-或C末端的几个残基的缺失或扩展不会影响观察到的单体变性曲线。该结果与我们先前的结构研究一致，我们的结构研究表明，成熟蛋白酶单体和单体前体的N-和C末端区域（其N-末端延伸超过成熟蛋白酶的延伸）在溶液中无序且灵活。与这些结果相反，活跃位点附近的SER-ALA取代提高了单体对尿素变性的稳定性。该观察结果被认为是由于涉及附近Leu Sidechains的ALA甲基的疏水堆积而产生的。单体结构特征的NMR测量正在与尿素变性谱一起使用，以阐明各种突变体的结构稳定关系。 我们继续研究构成蛋白酶同二聚体主要界面的N末端链和C末端链的动力学和相互作用。这项工作旨在进一步理解蛋白酶前体处理的机理。在源自X射线和NNMR工作的蛋白酶的静态结构模型中，来自两个单体的终端残基1-4和96-99形成了一个4链β-片（主要二聚体界面），其暴露自动解析易感循环包含残基5-9的易感性易感循环。然而，NMR横向自旋松弛（R2）分散数据，氢汇率和二维线形提供了有力的证据表明，在动态平衡中，残基1-9二聚体界面样品两个构型。数据提出的N末端的不稳定与先前关于各种蛋白酶突变体的报道中描述的结论一致，这表明涉及蛋白酶的溶剂裸露的N端链的相互作用在稳定同型二聚体的相互作用方面与涉及内部C-末端链链的相互作用相比，重要的是要重要得多。此外，提出的动态结构合理化了动力学数据，这些动力学数据表明N末端链折叠成蛋白酶前体的活性位点。这种构象开关允许未成熟N末端的分子间切割（GAG-POL处理的早期步骤）。最后，柔性N末端揭示了含有残基5-9的环的循环如何完全访问自动蛋白水解。 R2松弛分散曲线的分析原则上可以提供有关动态平衡中构象的化学移位和种群的定量信息，以及构象相互转换的速率。这些物理参数的准确值为表征与动态过程相关的结构和能量变化提供了基础。最近，我们表明，提取这些参数的最佳值及其不确定性需要仔细评估随机和系统的实验错误，以及正确应用统计标准以确保数据与适当的统计模型符合。当前的研究重点是分析由共同假设的动态参数中的误差分析，即经过交换的站点具有相同的固有放松率。我们进行了广泛的计算机模拟，为各种特定情况提供了此类错误的准确量值。此外，我们使用了雕刻师 - 刻痕方程来获得错误的一般理论表达式。理论方程式预测的结果与从数值计算中获得的结果非常吻合，并为光谱学家提供了简单的表达式，以估计在广泛的实验条件下误差的大小。