Agopian Audrey, Depollier Julien, Lionne Corinne, Divita Gilles
Centre de Recherches de Biochimie Macromoléculaire, Department of Molecular Biophysics and Therapeutic, FRE-2593 CNRS, 1919 Route de Mende, 34293 Montpellier, France.
J Mol Biol. 2007 Oct 12;373(1):127-40. doi: 10.1016/j.jmb.2007.07.044. Epub 2007 Aug 2.
Preventing dimerization of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) constitutes an alternative strategy to abolish virus proliferation. We have previously demonstrated that a short peptide derived from the Trp cluster of the connection domain disrupts the RT dimer by interacting with Trp24 and Phe61 in a cleft located between the fingers and the connection domains of p51. Both Trp24 and Phe61 of p51 are essential for the stability of the RT dimer. Here, in order to understand the requirement of Trp24 and Phe61 in the p66 subunit, we have investigated their implication in the formation of RT-primer/template (p/t) complexes and in RT processivity by combining pre-steady-state and steady-state kinetics with site-directed mutagenesis. We demonstrate that both residues are essential for proper binding of the p/t and control conformational changes required for RT ordered mechanism. Trp24 and Phe61 act on p/t binding and remodeling of the catalytic site. Phe61G mutation increases the binding "on" rate of both p/t and mismatched p/t, yielding an unfavorable RT-p/t for polymerase catalysis, unable to pursue mispair extension. Considering the structure of unliganded RT, Phe61 seems to be involved in the dynamics of p66 thumb-finger interactions and in stabilization of the p/t in the catalytic site. In contrast, the p66 Trp24G mutation alters the overall kinetics of p/t binding and is essentially involved in stabilizing the RT-p/t complex by contacting the 5' overhang of the template strand. Mutation of both Trp24 and Phe61 alters mispair extension efficiency, suggesting that disruption of the tight contacts between the fingers domain and the 5' overhang of the template strand increases RT fidelity and reduces RT processivity. Taken together, these studies infer that mutations altering the aromatic nature of Phe61 or Trp24 that may occur to counteract peptide inhibitors targeting this region will generate an unstable RT exhibiting low polymerase activity and higher fidelity. As such, our work suggests that the combined application of peptide-based RT dimerization inhibitors is likely to be highly efficient.
阻止人类免疫缺陷病毒1型逆转录酶（HIV-1 RT）二聚化是消除病毒增殖的一种替代策略。我们之前已经证明，源自连接结构域色氨酸簇的短肽通过与位于p51的手指结构域和连接结构域之间裂隙中的Trp24和Phe61相互作用来破坏RT二聚体。p51的Trp24和Phe61对于RT二聚体的稳定性都至关重要。在此，为了了解p66亚基中Trp24和Phe61的必要性，我们通过将预稳态和稳态动力学与定点诱变相结合，研究了它们在RT-引物/模板（p/t）复合物形成和RT持续合成能力中的作用。我们证明这两个残基对于p/t的正确结合以及RT有序机制所需的构象变化控制都是必不可少的。Trp24和Phe61作用于p/t结合以及催化位点的重塑。Phe61G突变增加了p/t和错配p/t的结合“开启”速率，产生了不利于聚合酶催化的RT-p/t，无法进行错配延伸。考虑到未结合配体的RT结构，Phe61似乎参与了p66拇指-手指相互作用的动力学以及催化位点中p/t的稳定。相比之下，p66 Trp24G突变改变了p/t结合的整体动力学，并且主要通过与模板链的5'突出端接触来稳定RT-p/t复合物。Trp24和Phe61两者的突变都会改变错配延伸效率，这表明手指结构域与模板链5'突出端之间紧密接触的破坏会提高RT保真度并降低RT持续合成能力。综上所述，这些研究推断，为了对抗靶向该区域的肽抑制剂而可能发生的改变Phe61或Trp24芳香性质的突变将产生一种不稳定的RT，其表现出低聚合酶活性和更高的保真度。因此，我们的工作表明基于肽的RT二聚化抑制剂的联合应用可能非常有效。
