Structure and functional implications of the polymerase active site region in a complex of HIV-1 RT with a double-stranded DNA template-primer and an antibody Fab fragment at 2.8 Å resolution
Structure and functional implications of the polymerase active site region in a complex of HIV-1 RT with a double-stranded DNA template-primer and an antibody Fab fragment at 2.8 Å resolution
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DOI：
10.1006/jmbi.1998.2208
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发表时间：
1998-12-11
影响因子：
5.6
通讯作者：
Arnold, E
中科院分区：
文献类型：
--
作者：
Ding, JP;Das, K;Arnold, E
用19-mer/18-mer双链DNA模板引物（dsDNA）和单克隆抗体28（Fab 28）的Fab片段复合的人免疫缺陷病毒1型（HIV-1）逆转录酶（RT）的结构在2.8埃分辨率下得到了改进。详细描述了聚合酶活性位点和邻近区域的结构，并提出了聚合酶催化和药物抑制机制的一些新见解。三个催化必需氨基酸残基（Asp 110、Asp 185和Asp 186）位于引物链的3'末端附近。引物末端的3 '-OH和Asp 185侧链之间的氢键的观察表明，Asp 185的羧酸酯可以作为聚合过程中引发亲核攻击的通用碱。几乎所有的蛋白质-DNA紧密相互作用都涉及核酸的糖-磷酸骨架的原子。然而，Tyr 183的苯氧基侧链是保守的YMDD基序的一部分，与第二双链体碱基对的核苷酸碱基具有氢键相互作用，并且预测在该位置处与所有Watson-Crick碱基对具有至少一个氢键。HIV-1 RT/dsDNA复合物中活性位点区域的结构与所有其他HIV-1 RT结构的比较表明，模板-引物结合伴随着YMDD基序的显著构象变化，这可能与非核苷抑制剂的聚合和抑制机制有关。“引物夹”（β 12-β 13发夹）与引物链3'末端的相互作用主要涉及Met 230和Gly 231的主链原子和引物末端磷酸。在不同的HIV-1 RT结构中观察到的引物夹的替代位置可能与DNA聚合和易位过程中通常发生的构象变化有关。在聚合酶活性位点附近，有许多芳香族残基参与能量上有利的π-π相互作用，并且可能参与催化过程的不同阶段之间的转换。主要负责模板-引物的精确定位的蛋白质结构元件（包括引物夹、模板夹和p66拇指的螺旋α H和α I）可以被认为起“易位轨道”的作用，其在聚合期间引导核酸和蛋白质的相对运动。(C)北京：科学出版社.
The structure of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) complexed with a 19-mer/18-mer double-stranded DNA template-primer (dsDNA) and the Fab fragment of monoclonal antibody 28 (Fab28) has been refined at 2.8 Angstrom resolution. The structures of the polymerase active site and neighboring regions are described in detail and a number of novel insights into mechanisms of polymerase catalysis and drug inhibition are presented. The three catalytically essential amino acid residues (Asp110, Asp185, and Asp186) are located dose to the 3' terminus of the primer strand. Observation of a hydrogen bond between the 3'-OH of the primer terminus and the side-chain of Asp185 suggests that the carboxylate of Asp185 could act as a general base in initiating the nucleophilic attack during polymerization. Nearly all of the close protein-DNA interactions involve atoms of the sugar-phosphate backbone of the nucleic acid. However, the phenoxyl side-chain of Tyr183, which is part of the conserved YMDD motif, has hydrogen-bonding interactions with nucleotide bases of the second duplex base-pair and is predicted to have at least one hydrogen bond with all Watson-Crick base-pairs at this position. Comparison of the structure of the active site region in the HIV-1 RT/dsDNA complex with all other HIV-1 RT structures suggests that template-primer binding is accompanied by significant conformational changes of the YMDD motif that may be relevant for mechanisms of both polymerization and inhibition by non-nucleoside inhibitors. interactions of the "primer grip" (the beta 12-beta 13 hairpin) with the 3' terminus of the primer strand primarily involve the main-chain atoms of Met230 and Gly231 and the primer terminal phosphate. Alternative positions of the primer grip observed in different HIV-1 RT structures may be related to conformational changes that normally occur during DNA polymerization and translocation. Ln the vicinity of the polymerase active site, there are a number of aromatic residues that are involved in energetically favorable pi-pi interactions and may be involved in the transitions between different stages of the catalytic process. The protein structural elements primarily responsible for precise positioning of the template-primer (including the primer grip, template grip, and helices alpha H and alpha I of the p66 thumb) can be thought of functioning as a "translocation track" that guides the relative movement of nucleic acid and protein during polymerization. (C) 1998 Academic Press.