Protein Structure Section, Macromolecular Crystallography Laboratory, National Cancer Institute at Frederick, Frederick, MD 21702, USA.
J Struct Biol. 2012 Mar;177(3):638-45. doi: 10.1016/j.jsb.2012.02.006. Epub 2012 Feb 16.
The ribonuclease H (RNase H) domain of retroviral reverse transcriptase (RT) plays a critical role in the life cycle by degrading the RNA strands of DNA/RNA hybrids. In addition, RNase H activity is required to precisely remove the RNA primers from nascent (-) and (+) strand DNA. We report here three crystal structures of the RNase H domain of xenotropic murine leukemia virus-related virus (XMRV) RT, namely (i) the previously identified construct from which helix C was deleted, (ii) the intact domain, and (iii) the intact domain complexed with an active site α-hydroxytropolone inhibitor. Enzymatic assays showed that the intact RNase H domain retained catalytic activity, whereas the variant lacking helix C was only marginally active, corroborating the importance of this helix for enzymatic activity. Modeling of the enzyme-substrate complex elucidated the essential role of helix C in binding a DNA/RNA hybrid and its likely mode of recognition. The crystal structure of the RNase H domain complexed with β-thujaplicinol clearly showed that coordination by two divalent cations mediates recognition of the inhibitor.
逆转录酶（RT）的核糖核酸酶 H（RNase H）结构域在生命周期中通过降解 DNA/RNA 杂合链中的 RNA 链发挥关键作用。此外，RNase H 活性对于从新生（-）和（+）链 DNA 中精确去除 RNA 引物是必需的。我们在此报告三种嗜性鼠白血病病毒相关病毒（XMRV） RT 的 RNase H 结构域的晶体结构，即（i）先前鉴定的缺失螺旋 C 的构建体，（ii）完整结构域，和（iii）与活性位点 α-羟基三酮抑制剂复合的完整结构域。酶促测定表明，完整的 RNase H 结构域保留了催化活性，而缺失螺旋 C 的变体仅具有轻微的活性，这证实了该螺旋对于酶活性的重要性。酶-底物复合物的建模阐明了螺旋 C 在结合 DNA/RNA 杂合链中的重要作用及其可能的识别模式。与 β-雪松醇结合的 RNase H 结构域的晶体结构清楚地表明，两个二价阳离子的协调介导了抑制剂的识别。
