Nmr Structural And Dynamics Studies Of Hiv-1 Protease

HIV-1 蛋白酶的核磁共振结构和动力学研究

• 批准号: 6966454
• 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/6966454
• 关键词: aspartic endopeptidases; conformation; enzyme inhibitors; human immunodeficiency virus 1; nuclear magnetic resonance spectroscopy; 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. Structural information has guided the design of the available anti-protease drugs. In a similar way knowledge of the monomer structure, as well as understanding of inter- and intra molecular interactions that affect dimer stability, should aid in the design of dimerization inhibitors. Therefore, we have determined the structure of the protease monomer. It 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. 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 NMR work, terminal residues 1-4 and 96-99 from both monomers form a 4-stranded b-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. The model discussed above was derived from data obtained for the protease bound to the inhibitor DMP323. The free protease exhibits R2 dispersion that is qualitatively similar to that obtained for the protease/DMP323 complex. However, the amplitude of the R2 dispersion is significantly smaller in the case of the free protease. Work is currently underway to find the reason for the quantitative difference in relaxation dispersion observed between free and ligand-bound proteases. 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. However , 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. We have developed a systematic procedure for obtaining best-fitted values of the desired physical parameters and their uncertainties. We show that a judicious choice of initial fitting parameters is required in order that the final fitting parameters correspond the global, rather than local, chi square minimum in the parameter space. In addition, we show that when exchange is outside the fast limit for some sites, robust parameter estimates are obtained by simultaneously fitting 1H and 15N R2 dispersion data of multiple residues in a protein. In contrast, when exchange is in the fast limit for all sites, uncertainties in populations and chemical shifts are large and correlated. This problem can be often be over come by recording data at lower temperature and/or at larger external field, so that exchange is not in the fast limit at all sites.

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。 结构信息指导了可用的抗癌药物的设计。以类似的方式了解单体结构，以及对影响二聚体稳定性的分子间相互作用的理解，应有助于设计二聚化抑制剂。因此，我们确定了蛋白酶单体的结构。它包含一个折叠的核心域，该结构域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。 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NMR工作的蛋白酶的静态结构模型中，两个单体的末端残基1-4和96-99形成了4链B-片（主要的二聚体界面），裸露的自动溶解易感循环包含残基5-9，并连接到该残基。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。 上面讨论的模型是从与抑制剂DMP323结合的蛋白酶获得的数据得出的。游离蛋白酶表现出与蛋白酶/DMP323复合物获得的R2分散剂。但是，在游离蛋白酶的情况下，R2色散的幅度明显较小。目前正在进行工作，以找到在自由和结合配体蛋白酶之间观察到的弛豫分散剂定量差异的原因。 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。但是，提取这些参数的最佳值及其不确定性需要仔细评估实验错误（随机和系统的）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。我们已经开发了一个系统的程序，用于获得所需的物理参数及其不确定性的最合适的值。我们表明，为了使最终拟合参数对应于全局，而不是本地的CHI Square在参数空间中，则需要明智地选择初始拟合参数。此外，我们表明，当交换在某些站点的快速限制之外时，通过同时拟合1H和15N R2分散数据的多个残基的蛋白质中的稳健参数估计值。相比之下，当交换在所有站点的快速限制中时，人群和化学位移的不确定性都大且相关。在较低的温度和/或较大的外部场上记录数据通常可以解决这个问题，以便在所有站点的快速限制中交换。