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首页> 外文期刊>Biochemistry >The #beta#7-#beta#8 Loop of the p51 Subunit in the Heterodimeric (p66/p51) Human Immunodeficiency Virus Type 1 Reverse Transcriptase Is Essential for the Catalytic Function of the p66 Subunit
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The #beta#7-#beta#8 Loop of the p51 Subunit in the Heterodimeric (p66/p51) Human Immunodeficiency Virus Type 1 Reverse Transcriptase Is Essential for the Catalytic Function of the p66 Subunit

机译：异二聚体（p66 / p51）人类免疫缺陷病毒1型逆转录酶中p51亚基的＃beta＃7-＃beta＃8环对于p66亚基的催化功能至关重要

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摘要

The heterodimeric human immunodeficiency virus type 1 reverse transcriptase (HIV -1 RT) is composed of p66 and p51 subunits, p66 being the catalytic subunit. Our earlier investigation on the role of p51 in the catalytic process has shown that the p51 subunit facilitates the loading of the p66 subunit onto the template primer (TP). We had postulated that the #beta#7-#beta#8 loop of the p51 subunit may be involved in opening the polymerase cleft of p66 for DNA binding [Pandey, v. N., et al. (1996) Biochemistry 35, 2168]. We report here that deletion or alanine substitution of four residues of the #beta#7-#beta#8 loop results in severe impairment of the polymerase function of the heterodimeric enzyme. The enzyme activity was restored to the wild-type levels when the mutant p66 subunit was dimerized with the wild-type p51, suggesting that the intact #beta#7-#beta#8 loop in the p51 subunit is indispensable for the catalytic function of p66. Further, the template primer binding ability of the enzyme was significantly reduced upon deletion or alanine substitution in the #beta#7-#beta#8 loop. Interestingly, the loss of the TP binding ability of the mutant p66 was restored upon dimerization with wild-type p51. Examination of the glycerol gradient ultracentrifugation analysis revealed that while the wild-type HIV -1 RT sediments as a dimeric protein, the mutant enzymes carrying deletion or alanine substitution in both the subunits sediment predominantly as monomeric proteins, suggesting their inability to form stable dimers. In contrast, mutant p66 dimerized with wild-type p51 (p66f./p51WT and p66Ala/p51WT) sedimented at the dimeric position. Taken together, these results clearly implicate the importance of the #beta#7 -#beta#8 loop of p51 in the formation of stable functional heterodimers.
机译：异二聚体人类免疫缺陷病毒1型逆转录酶（HIV -1 RT）由p66和p51亚基组成，p66是催化亚基。我们对p51在催化过程中的作用的较早研究表明，p51亚基可促进p66亚基加载到模板引物（TP）上。我们已经假定p51亚基的＃beta＃7-＃beta＃8环可能参与打开p66的聚合酶裂口以进行DNA结合[Pandey，v.N.，et al。等。 （1996）生物化学35，2168]。我们在这里报告的＃beta＃7-＃beta＃8环的四个残基的删除或丙氨酸取代导致异二聚酶的聚合酶功能严重受损。当突变体p66亚基与野生型p51二聚化时，酶活性恢复到野生型水平，这表明p51亚基中完整的＃beta＃7-＃beta＃8环对于P53的催化功能是必不可少的。 66。此外，在＃beta＃7-＃beta＃8环中缺失或丙氨酸取代后，酶的模板引物结合能力显着降低。有趣的是，突变体p66的TP结合能力的丧失在与野生型p51二聚化后得以恢复。甘油梯度超速离心分析的检查表明，虽然野生型HIV -1 RT沉淀为二聚体蛋白，但两个亚基中都带有缺失或丙氨酸取代的突变酶主要沉淀为单体蛋白，表明它们无法形成稳定的二聚体。相反，突变体p66与野生型p51（p66f./p51WT和p66Ala / p51WT）二聚化，沉淀在二聚体位置。综上所述，这些结果清楚地暗示了p51的＃beta＃7-＃beta＃8环在形成稳定功能性异二聚体中的重要性。

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