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: 6814480
• 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/6814480
• 关键词: aspartic endopeptidases; chemical stability; enzyme activity; enzyme complex; enzyme inhibitors; enzyme mechanism; enzyme structure; human immunodeficiency virus 1; intermolecular interaction; nuclear magnetic resonance spectroscopy; virus protein

The active HIV-1 protease is a homodimer made up of monomers containing 99 amino acid residues. All drugs directed against the protease have targeted the substrate-binding site of the dimer. Although at least six drugs have been developed that binding tightly to the active site, one unfortunate consequence of targeting a single drug binding site on the protease has been the emergence of viral strains which carry multi-drug resistant mutations on their protease constructs. For this reason, there is considerable interest in identifying other protease sites that are suitable drug targets. Although the protease monomer is completely inactive, we have demonstrated that it is folded and therefore contains numerous potential alternative drug-binding sites, making it an attractive anti-HIV target. In order to overcome problems with monomer aggregation, we have designed proteins in which the N- and C-terminal regions are linked by intra-monomer disulfide bonds. In particular, cysteine residues were introduced at positions 2 and at either 97 or 98 of the protease amino acid sequence and using NMR have shown that the Q2C/L97C monomer construct exhibits a fold similar to that observed for the monomer subunit in the active dimer. It is anticipated that monomeric proteases of this kind will aid in the discovery of novel inhibitors, that bind to the monomer at the dimerization interface, rather than at the active site. Such inhibitors could circumvent the problem of multidrug-resistance invariably observed with current HIV-1 protease drugs all of which bind at the active site. We have extend thses studies of the protease monomer by using NMR to determine the first solution structure of a protease monomer, the HIV-1 protease spanning the region Phe1-Ala95 (PR1-95). Except for the terminal regions (residues 1-10 and 91-95) that are disordered, the tertiary fold of the remainder of the protease monomer is essentially identical to that of the individual subunit of the active protease dimer. In the monomer, the side chains of buried residues stabilizing the active site interface in the dimer, such as Asp25, Asp29, and Arg87, are now exposed to solvent. The flap dynamics in the monomer are similar to that of the free protease dimer. We also show that the protease domain of an optimized precursor flanked by 56 amino acids of the N-terminal transframe region is predominantly monomeric exhibiting a tertiary fold that is quite similar to the PR1-95 structure. This explains the very low catalytic activity observed for the protease prior to its maturation at its N-terminus as compared to the mature protease, which is an active stable dimer under identical conditions. An addition of even 2 amino acids to the N-terminus of the mature protease significantly increases its dissociation into monomer. Knowledge of the protease monomer structure and critical features of its dimerization may aid in the screening and design of compounds that target the protease prior to its maturation from the Gag-Pol precursor. We have advanced NMR methodology for studying slow protein motions (on the millesecond to microsecond timescale) in solution by developing pulses sequences that measure the relaxation dispersion of amide proton and backbone carbonyl spins in proteins. These measurements together with measurements of NMR lineshapes, carried out at temperatures ranging from 2 to 28 ?C, and measurements of hydrogen-deuterium exchange are being analyzed with the goal of developing a detailed model of the conformational fluctuations that occur at the dimer interface of the protease, both when it is free and when it is bound to a potent inhibitor.

活性HIV-1蛋白酶是由包含99个氨基酸残基的单体组成的同型二聚体。针对蛋白酶的所有药物都针对二聚体的底物结合位点。尽管已经开发了与活性位点紧密结合的至少六种药物，但在蛋白酶上靶向单个药物结合位点的不幸后果是病毒菌株的出现，病毒菌株在其蛋白酶构建体上携带多药物抗性突变。因此，识别其他是合适的药物靶标的其他蛋白酶位点具有很大的兴趣。尽管蛋白酶单体完全不活跃，但我们已经证明了蛋白酶单体已折叠，因此包含许多潜在的替代药物结合位点，使其成为有吸引力的抗HIV靶标。为了克服单体聚集问题，我们设计了蛋白质，其中N-和C末端区域与知识内二硫键键关联。特别是，在位置2和蛋白酶氨基酸序列的97或98处引入半胱氨酸残基，并使用NMR引入了Q2C/L97C单体构建体表现出类似于活性二聚体中单体亚基的倍数。可以预料，这种类型的单体蛋白酶将有助于发现新型抑制剂，这些抑制剂与二聚化界面的单体结合，而不是在活性位点结合。这种抑制剂可以避免使用当前的HIV-1蛋白酶药物观察到多药抗性的问题，所有这些药物均在活性位点结合。 我们通过使用NMR来确定蛋白酶单体的第一个溶液结构，即横跨PHE1-ALA95区域的HIV-1蛋白酶（PR1-95）来扩展对蛋白酶单体的ThSE研究。除了无序的末端区域（残基1-10和91-95）外，其余蛋白酶单体的第三折与活性蛋白酶二聚体的单个亚基的第三折基本相同。在单体中，掩埋残留物的侧链稳定二聚体中的活性位点界面，例如ASP25，ASP29和ARG87，现在暴露于溶剂中。单体中的瓣动力学与游离蛋白酶二聚体的瓣动力学相似。我们还表明，N末端转框区域的56个氨基酸的优化前体的蛋白酶结构域主要是单体，表现出与PR1-95结构非常相似的三级褶皱。这解释了与成熟蛋白酶相比，在其N末端成熟之前观察到的蛋白酶在其成熟之前观察到的非常低的催化活性，这在相同条件下是一种活跃的稳定二聚体。在成熟蛋白酶的N末端增加了2个氨基酸，显着增加了其分离为单体。了解蛋白酶单体结构及其二聚化的关键特征可能有助于筛选和设计靶向蛋白酶在其从GAG-POL前体成熟之前的化合物。 我们通过开发脉冲序列来测量蛋白质中酰胺质子和骨链羰基旋转的脉冲序列来研究溶液中的慢速蛋白运动（在毫秒至微秒的时间尺度上）的高级NMR方法。这些测量以及NMR线路的测量值在2至28 c的温度下进行，并且正在分析氢 - 偏见交换的测量，目的是开发出在蛋白酶dimease界面上发生的详细构型波动模型，两者是免费的，并且在蛋白酶的二聚体界面上，这两者是免费的。