The mobility of an HIV-1 integrase active site loop is correlated with catalytic activity
The mobility of an HIV-1 integrase active site loop is correlated with catalytic activity
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DOI：
10.1021/bi9907173
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发表时间：
1999-07-13
期刊：
影响因子：
2.9
通讯作者：
Choe, S
中科院分区：
文献类型：
--
作者：
Greenwald, J;Le, V;Choe, S
HIV-1的复制需要病毒cDNA在病毒编码整合酶蛋白的指导下与宿主染色体DNA共价整合。在这里，我们利用蛋白质工程和x射线晶体学探讨了在整合酶活性位点附近的蛋白质表面环的重要性。我们重新确定了整合酶催化结构域（残基50-212）的结构，使用独立的相集在1.7埃分辨率。该结构在其n端延伸螺旋α 4（残基149-154），从而确定了三个保守活性位点残基的位置。在这个和以前的结构中，很明显是由残基141-148组成的构象柔性环。为了探索柔韧性在这个循环中的作用，我们用Ala残基替换了Gly 140和Gly 149，这些残基似乎起着构象铰链的作用。催化结构域突变体G149A和G140A/G149A的x射线结构显示α 4和相邻环的进一步刚性。体外活性测定显示，这些突变体的催化功能受损。然而，通过紫外交联检测，这些突变体对DNA结合亲和力的影响最小。我们认为这种活性位点环的构象灵活性对于结合后催化步骤是重要的。
Replication of HIV-1 requires the covalent integration of the viral cDNA into the host chromosomal DNA directed by the virus-encoded integrase protein. Here we explore the importance of a protein surface loop near the integrase active site using protein engineering and X-ray crystallography. We have redetermined the structure of the integrase catalytic domain (residues 50-212) using an independent phase set at 1.7 Angstrom resolution. The structure extends helix alpha 4 on its N-terminal side (residues 149-154), thus defining the position of the three conserved active site residues. Evident in this and in previous structures is a conformationally flexible loop composed of residues 141-148. To probe the role of flexibility in this loop, we replaced Gly 140 and Gly 149, residues that appear to act as conformational hinges, with Ala residues. X-ray structures of the catalytic domain mutants G149A and G140A/G149A show further rigidity of alpha 4 and the adjoining loop. Activity assays in vitro revealed that these mutants are impaired in catalysis. The DNA binding affinity, however, is minimally affected by these mutants as assayed by UV cross-linking. We propose that the conformational flexibility of this active site loop: is important for a postbinding catalytic step.