The β1'-β2' Motif of the RNase H Domain of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Is Responsible for Conferring Open Conformation to the p66 Subunit by Displacing the Connection Domain from the Polymerase Cleft.
The β1'-β2' Motif of the RNase H Domain of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Is Responsible for Conferring Open Conformation to the p66 Subunit by Displacing the Connection Domain from the Polymerase Cleft.
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人类免疫缺陷病毒 1 型逆转录酶的 RNase H 结构域的 β1-β2 基序负责通过置换聚合酶裂口的连接结构域来赋予 p66 亚基开放构象。
DOI：
10.1021/acs.biochem.7b00005
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发表时间：
2017
期刊：
影响因子：
2.9
通讯作者：
Pandey,VirendraN
中科院分区：
文献类型：
--
作者：
Pandey,AshutoshK;Dixit,Updesh;Kholodovych,Vlad;Comollo,ThomasW;Pandey,VirendraN
异二聚体人类免疫缺陷病毒1型逆转录酶由p66和p51亚基组成。而在 p51 亚基中，连接域隐藏在聚合酶裂缝中；它被有效地从催化活性 p66 亚基的裂缝中取代。连接域是如何从 p66 聚合酶裂口重新定位的？ RNase H 结构域在此过程中发挥作用吗？为了回答这个问题，我们通过逐步添加 RNase H 结构域的 N 端基序来延伸 p51 的 C 端区域，以生成 p54、p57、p60 和 p63 衍生物。我们发现p51的所有C端延伸衍生物均呈现开放构象，与模板引物结合并催化聚合酶反应。甘油梯度超速离心分析表明，只有p54以单体形式沉淀，而其他衍生物均呈同二聚体构象。我们提出了一个模型来解释催化活性 p54 衍生物的单体构象，该衍生物携带来自 RNase H 结构域的额外 21 个残基长 β1'−β2' 基序。我们的结果表明，RNase H 结构域的 β1'-β2' 基序可能负责将连接结构域从假定的单体 p66 的聚合酶裂缝中置换出来。然后，不稳定的伸长的 p66 分子可以很容易地与 p51 二聚化，以呈现稳定的二聚体构象。
The heterodimeric human immunodeficiency virus type 1 reverse transcriptase is composed of p66 and p51 subunits. While in the p51 subunit, the connection domain is tucked in the polymerase cleft; it is effectively displaced from the cleft of the catalytically active p66 subunit. How is the connection domain relocated from the polymerase cleft of p66? Does the RNase H domain have any role in this process? To answer this question, we extended the C-terminal region of p51 by stepwise addition of N-terminal motifs of RNase H domain to generate p54, p57, p60, and p63 derivatives. We found all of the C-terminal extended derivatives of p51 assume open conformation, bind to the template-primer, and catalyze the polymerase reaction. Glycerol gradient ultracentrifugation analysis showed that only p54 sedimented as a monomer, while other derivatives were in a homodimeric conformation. We proposed a model to explain the monomeric conformation of catalytically active p54 derivative carrying additional 21-residues long β1′−β2′ motif from the RNase H domain. Our results indicate that the β1′-β2′ motif of the RNase H domain may be responsible for displacing the connection domain from the polymerase cleft of putative monomeric p66. The unstable elongated p66 molecule may then readily dimerize with p51 to assume a stable dimeric conformation.