Foulkes Jennifer E, Prabu-Jeyabalan Moses, Cooper Deyna, Henderson Gavin J, Harris Janera, Swanstrom Ronald, Schiffer Celia A
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 364 Plantation Street, Worcester, 01605, USA.
J Virol. 2006 Jul;80(14):6906-16. doi: 10.1128/JVI.01900-05.
Sequence variability associated with human immunodeficiency virus type 1 (HIV-1) is useful for inferring structural and/or functional constraints at specific residues within the viral protease. Positions that are invariant even in the presence of drug selection define critically important residues for protease function. While the importance of conserved active-site residues is easily understood, the role of other invariant residues is not. This work focuses on invariant Thr80 at the apex of the P1 loop of HIV-1, HIV-2, and simian immunodeficiency virus protease. In a previous study, we postulated, on the basis of a molecular dynamics simulation of the unliganded protease, that Thr80 may play a role in the mobility of the flaps of protease. In the present study, both experimental and computational methods were used to study the role of Thr80 in HIV protease. Three protease variants (T80V, T80N, and T80S) were examined for changes in structure, dynamics, enzymatic activity, affinity for protease inhibitors, and viral infectivity. While all three variants were structurally similar to the wild type, only T80S was functionally similar. Both T80V and T80N had decreased the affinity for saquinavir. T80V significantly decreased the ability of the enzyme to cleave a peptide substrate but maintained infectivity, while T80N abolished both activity and viral infectivity. Additionally, T80N decreased the conformational flexibility of the flap region, as observed by simulations of molecular dynamics. Taken together, these data indicate that HIV-1 protease functions best when residue 80 is a small polar residue and that mutations to other amino acids significantly impair enzyme function, possibly by affecting the flexibility of the flap domain.
与人类免疫缺陷病毒1型（HIV-1）相关的序列变异性有助于推断病毒蛋白酶内特定残基的结构和/或功能限制。即使在药物选择存在的情况下仍保持不变的位置定义了蛋白酶功能至关重要的残基。虽然保守的活性位点残基的重要性很容易理解，但其他不变残基的作用却并非如此。这项工作聚焦于HIV-1、HIV-2和猿猴免疫缺陷病毒蛋白酶P1环顶端的不变苏氨酸80。在先前的一项研究中，我们基于未结合配体的蛋白酶的分子动力学模拟推测，苏氨酸80可能在蛋白酶瓣片的移动性中发挥作用。在本研究中，实验和计算方法都被用于研究苏氨酸80在HIV蛋白酶中的作用。研究了三种蛋白酶变体（T80V、T80N和T80S）在结构、动力学、酶活性、对蛋白酶抑制剂的亲和力以及病毒感染性方面的变化。虽然所有三种变体在结构上都与野生型相似，但只有T80S在功能上相似。T80V和T80N对沙奎那韦的亲和力均降低。T80V显著降低了酶切割肽底物的能力，但保持了感染性，而T80N则消除了活性和病毒感染性。此外，如分子动力学模拟所观察到的，T80N降低了瓣片区域的构象灵活性。综上所述，这些数据表明，当残基80为小极性残基时，HIV-1蛋白酶功能最佳，而突变为其他氨基酸可能会通过影响瓣片结构域的灵活性而显著损害酶的功能。
