Efavirenz binding to HIV-1 reverse transcriptase monomers and dimers.
Efavirenz binding to HIV-1 reverse transcriptase monomers and dimers.
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DOI：
10.1021/bi901579y
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发表时间：
2010-01-26
期刊：
影响因子：
2.9
通讯作者：
Barkley, Mary D.
中科院分区：
文献类型：
--
作者：
Braz, Valerie A.;Holladay, Leslie A.;Barkley, Mary D.
EFV是HIV-1逆转录酶(RT)的非核苷类逆转录酶抑制剂，用于治疗艾滋病。RT是由p66和p51亚基组成的异源二聚体，p51是由p66经HIV酶C端截短而产生的。单体可以形成p66/p66和p51/p51同源二聚体，也可以形成p66/p51杂二聚体。二聚化和Eefavirenz结合是耦合过程。在p66/p51-EFV复合体的晶体结构中，药物与p66亚单位结合。通过平衡透析、色氨酸荧光和天然凝胶电泳法研究了Eefavirenz与野生型和二聚化缺陷RT蛋白的结合。单体和同源二聚体的结合化学计量比均为1：1。P66和p51-EFV络合物的平衡解离常数均为~2.5μM，p66/P66-EFV络合物的平衡解离常数为250 nM，p51/p51-EFV络合物的平衡解离常数为7 nM。根据二聚反应与缓蚀剂结合的热力学联系计算出p66/p51-EFV络合物的平衡离解常数为92 nM。荧光监测的结合和解离动力学较慢。过程曲线分析表明，单体和杂二聚体的缔合速率常数k_1~13.5M~(-1)·S~(-1)和单体的离解速率常数k~(-1)~1×10~(-4)S~(-1)为一步直接键合机理。提出了一种构象选择机制来解释缔合率低的原因。这些结果表明，Eefavirenz是一种缓慢的紧密结合的抑制剂，能够结合所有形式的RT，并表明NNRTI在单体和二聚体中的结合部位是相似的。
Efavirenz (EFV) is a nonnucleoside reverse transcriptase inhibitor (NNRTI) of HIV-1 reverse transcriptase (RT) used for the treatment of AIDS. RT is a heterodimer composed of p66 and p51 subunits; p51 is produced from p66 by C-terminal truncation by HIV protease. The monomers can form p66/p66 and p51/p51 homodimers as well as p66/p51 heterodimer. Dimerization and efavirenz binding are coupled processes. In the crystal structure of the p66/p51—EFV complex, the drug is bound to the p66 subunit. The binding of efavirenz to wild-type and dimerization-defective RT proteins was studied by equilibrium dialysis, tryptophan fluorescence and native gel electrophoresis. A 1:1 binding stoichiometry was determined for both monomers and homodimers. Equilibrium dissociation constants are ~2.5 μM for both p66— and p51—EFV complexes, 250 nM for p66/p66—EFV complex, and 7 nM for p51/p51—EFV complex. An equilibrium dissociation constant of 92 nM for p66/p51—EFV complex was calculated from the thermodynamic linkage between dimerization and inhibitor binding. Binding and unbinding kinetics monitored by fluorescence were slow. Progress curve analyses revealed a one-step, direct binding mechanism with association rate constants k1 ~13.5 M–1 s–1 for monomers and heterodimer and dissociation rate constants k–1 ~1 × 10–4 s–1 for monomers. A conformational selection mechanism is proposed to account for the slow association rate. These results show that efavirenz is a slow, tight-binding inhibitor capable of binding all forms of RT and suggest that the NNRTI binding site in monomers and dimers is similar.
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影响因子：
2.9
作者：
Braz VA;Howard KJ
通讯作者：
Howard KJ
影响因子：
6.7
作者：
Figueiredo A;Moore KL;Mak J;Sluis-Cremer N;de Bethune MP;Tachedjian G
通讯作者：
Tachedjian G
影响因子：
2.9
作者：
Ghosh, M;Jacques, PS;leGrice, SFJ
通讯作者：
leGrice, SFJ
影响因子：
2.9
作者：
Ignatov, ME;Berdis, AJ;Barkley, MD
通讯作者：
Barkley, MD
影响因子：
3.5
作者：
RESTLE, T;MULLER, B;GOODY, RS
通讯作者：
GOODY, RS