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: 6104608
• 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/6104608
• 关键词: chemical stability; drug design /synthesis /production; endopeptidases; enzyme activity; enzyme complex; enzyme mechanism; enzyme structure; human immunodeficiency virus 1; intermolecular interaction; nuclear magnetic resonance spectroscopy; protease inhibitor; protein structure function; solutions; virulence; virus protein

Our recent work has focused upon studies of the internal motions of the HIV-1 protease (a) complexed with the potent inhibitor, DMP323, a member of a novel class of symmetric, specific and potent (Ki ~ 10-1000 pM) inhibitors in which a diol moiety is incorporated into a seven membered cyclic urea ring and (b) free in solution, as a fully active, but stable protease mutant (Q7K, L33I, L63I). The studies of the protease/DMP323 complex represent a significant extension of previous work that was made possible by our development of a novel method to study slow backbone motions in the protease, using complementary measurements of amide 15N and 1H transverse relaxation times. For this purpose we use a protein which is highly deuterated at non-exchangable hydrogen sites, and is dissolved in H2O so that the exchangable amide hydrogens are protonated. The high level of deuteration minimizes the contribution of 1H-1H dipolar interactions to the proton relaxation rate. This in turn facilitates the detection of the conformational exchange contribution to the proton relaxation rate. Measuring both the 1H and 15N transverse relaxation rates significantly increases the likely hood that relaxation due to slow conformational exchange will be detected. The new experiments clearly reveal the contribution of conformational exchange to the proton relaxation rates of residues T4, L5 and W6 in the autolysis sensitive loop. Previously, these slow motions were indirectly inferred from the absence of the L5 NH signal in our spectra. The new experiments also revealed that in the free protease, residues G48 through I54 (in the flaps of the protein) undergo slow conformation exchange. These results suggest that the flaps act as gates that open and close on the timescale of milleseconds, permitting entry and exit of ligands from the active site of the enzyme.

我们最近的工作集中于对 HIV-1蛋白酶的内部运动（a）与 有效的抑制剂DMP323，新型对称类的成员， 特异性和有效（KI〜10-1000 pm）抑制剂，其中二醇 将部分合并到一个七个成员的环状尿素环中，然后 （b）在溶液中免费作为一个完全活跃但稳定的蛋白酶突变体 （Q7K，L33i，L63i）。蛋白酶/DMP323复合物的研究 代表了先前工作的重大扩展 通过我们开发一种新的方法来研究慢的方法 蛋白酶中的骨干运动，使用互补 酰胺15N和1H横向松弛时间的测量。 为此，我们使用了高度一个高剥离的蛋白质 不可交换的氢位点，并溶解在H2O中，以便 交换酰胺氢质子化。高水平 申请最小化1H-1H偶极的贡献 与质子松弛率的相互作用。这又促进了 检测构象交换对 质子松弛率。测量1H和15N横向 放松率显着增加了可能的引擎盖 将检测到由于缓慢的构象交换而引起的放松。 新实验清楚地揭示了 与残基的质子松弛率的构象交换 自溶解敏感环中的T4，L5和W6。以前，这些 从没有L5的情况下间接推断出缓慢的运动 NH信号在我们的光谱中。新实验还显示 自由蛋白酶，残留G48至i54（在 蛋白质）进行缓慢的构象交换。这些结果 建议襟翼充当开放和关闭的门 米尔秒的时间尺度，允许进入并退出配体 酶的活性部位。