IDENTIFICATION OF CONSERVED AMINO-ACID-RESIDUES CRITICAL FOR HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 INTEGRASE FUNCTION-INVITRO
IDENTIFICATION OF CONSERVED AMINO-ACID-RESIDUES CRITICAL FOR HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 INTEGRASE FUNCTION-INVITRO
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DOI：
10.1128/jvi.66.11.6361-6369.1992
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发表时间：
1992-11-01
影响因子：
5.4
通讯作者：
CRAIGIE, R
中科院分区：
文献类型：
--
作者：
ENGELMAN, A;CRAIGIE, R
我们已经探讨了有限的蛋白水解和功能组织的定点诱变选定的氨基酸残基的人类免疫缺陷病毒1型整合酶蛋白的结构组织。蛋白质的中心区域对蛋白水解具有相对抗性。纯化在该区域或在蛋白质的N-末端部分（包括推定的锌结合基序）具有改变的氨基酸的蛋白质，并测定其3'加工、DNA链转移和体外崩解活性。一般而言，这些突变对3'加工和DNA链转移具有平行影响，表明整合酶可能利用单个活性位点进行两种反应。在所有三种检测中完全失活的蛋白质中，仅有的蛋白质在中心区域的保守氨基酸处含有突变，这表明蛋白质的这一部分可能参与催化。相比之下，没有一个在N-末端区域的突变导致在所有三种测定中无活性的蛋白质，这表明整合酶的这一部分可能不是催化所必需的。崩解反应对这些氨基酸取代特别不敏感，表明对于3'加工和DNA链转移重要的一些功能可能对于崩解不敏感。
We have probed the structural organization of the human immunodeficiency virus type 1 integrase protein by limited proteolysis and the functional organization by site-directed mutagenesis of selected amino acid residues. A central region of the protein was relatively resistant to proteolysis. Proteins with altered amino acids in this region, or in the N-terminal part of the protein that includes a putative zinc-binding motif, were purified and assayed for 3' processing, DNA strand transfer, and disintegration activities in vitro. In general, these mutations had parallel effects on 3' processing and DNA strand transfer, suggesting that integrase may utilize a single active site for both reactions. The only proteins that were completely inactive in all three assays contained mutations at conserved amino acids in the central region, suggesting that this part of the protein may be involved in catalysis. In contrast, none of the mutations in the N-terminal region resulted in a protein that was inactive in all three assays, suggesting that this part of integrase may not be essential for catalysis. The disintegration reaction was particularly insensitive to these amino acid substitutions, indicating that some function that is important for 3' processing and DNA strand transfer may be dispensable for disintegration.