Role of the ''helix clamp'' in HIV-1 reverse transcriptase catalytic cycling as revealed by alanine-scanning mutagenesis
Role of the ''helix clamp'' in HIV-1 reverse transcriptase catalytic cycling as revealed by alanine-scanning mutagenesis
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DOI：
10.1074/jbc.271.21.12213
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发表时间：
1996-05-24
影响因子：
4.8
通讯作者：
Wilson, SH
中科院分区：
文献类型：
--
作者：
Beard, WA;Minnick, DT;Wilson, SH
HIV-1 逆转录酶的残基 259-284 表现出与其他核酸聚合酶的序列同源性，并被称为“螺旋夹”（Hermann, T.、Meier, T.、Gotte, M. 和 Heumann, H. (1994) Nucleic Acids Res, 22, 4625-4633），因为晶体学证据表明这些残基是两个残基的一部分。与 DNA 相互作用的 α 螺旋（α H 和 α I）。丙氨酸扫描诱变先前已证明 α H 中的几个残基与核酸产生重要的相互作用并影响移码保真度。为了确定 α I（残基 278-286）在催化循环过程中的作用，我们通过将每个残基一一改变为丙氨酸，进行了从位置 277 到位置 287 的系统定点诱变。每个突变蛋白均得到表达，并且除了 L283A 和 T286A 之外，都是可溶的。对可溶性突变酶进行纯化和表征，与α H 的丙氨酸突变体相比，α I 中的丙氨酸取代对模板没有显着影响。通过对 K-m,K-T 缺乏影响表明引物 (T.P) 结合。 P、K-i 表示 3'-叠氮基-2',3'-二脱氧胸苷 5'-三磷酸、k(off,T.P) 和持续合成能力。与这些观察结果一致，突变酶的保真度没有受到影响。然而，相对于野生型酶，α I 的丙氨酸诱变降低了每个突变体的表观活性，用T滴定显示出最低活性的两个突变体。 P（L282A 和 R284A）证明这些突变酶可以结合 T 。相反，从“爆发”实验确定的活性位点浓度表明，较低的活性是由于与 T 有效结合的酶群体较少。 P. 螺旋夹的基本侧链和 DNA 主链之间假定的静电相互作用要么非常弱，要么动力学沉默。相反，α H 的几个残基和 DNA 小沟（3'-引物末端的 3-5 个核苷酸）之间的相互作用被认为对于 DNA 结合和保真度至关重要。
Residues 259-284 of HIV-1 reverse transcriptase exhibit sequence homology with other nucleic acid polymerases and have been termed the ''helix clamp'' (Hermann, T., Meier, T., Gotte, M., and Heumann, H. (1994) Nucleic Acids Res, 22, 4625-4633), since crystallographic evidence indicates these residues are part of two alpha-helices (alpha H and alpha I) that interact with DNA. Alanine-scanning mutagenesis has previously demonstrated that several residues in alpha H make important interactions with nucleic acid and influence frameshift fidelity. To define the role of alpha I (residues 278-286) during catalytic cycling, we performed systematic site directed mutagenesis from position 277 through position 287 by changing each residue, one by one, to alanine, Each mutant protein was expressed and, except for L283A and T286A, was soluble. The soluble mutant enzymes were purified and characterized, In contrast to alanine mutants of alpha H, alanine substitution in alpha I did not have a significant effect on template . primer (T . P) binding as revealed by a lack of an effect on K-m,K-T . P, K-i for 3'-azido-2',3'-dideoxythymidine 5'-triphosphate, k(off,T . P), and processivity. Consistent with these observations, the fidelity of the mutant enzymes was not influenced. However, alanine mutagenesis of alpha I lowered the apparent activity of every mutant relative to wild-type enzyme, Titration of two mutants exhibiting the lowest activity with T . P (L282A and R284A) demonstrated that these mutant enzymes could bind T . P stoichiometrically and tightly, In contrast, active site concentrations determined from ''burst'' experiments suggest that the lower activity is due to a smaller population of enzyme bound productively to T . P. The putative electrostatic interactions between the basic side chains of the helix clamp and the DNA backbone are either very weak or kinetically silent, In contrast, interactions between several residues of alpha H and the DNA minor groove, 3-5 nucleotides from the 3'-primer terminus, are suggested to be critical for DNA binding and fidelity.