The configurational space sampled by the finger and thumb subdomains of the p66 subunit of HIV-1 reverse transcriptase was investigated by Q-band double electron–electron resonance pulsed electron paramagnetic resonance spectroscopy, a method for determining long-range distances between pairs of nitroxide spin-labels introduced via surface-engineered cysteine residues. Four constructs were examined, each containing two spin-labels in the p66 subunit, one in the finger subdomain and the other in the thumb subdomain. In the unliganded state, open and closed configurations for the finger and thumb subdomains are observed with the distribution between these states modulated by the spin-labels and associated mutations, in contrast to crystallographic data in which the unliganded state crystallizes in the closed conformation. Upon addition of double-stranded DNA, all constructs adopt open conformations consistent with previous crystallographic data in which the position of the thumb and finger subdomains is determined by contacts with the bound oligonucleotide duplex (DNA or DNA/RNA). Likewise, binary complexes with five different non-nucleoside reverse transcriptase inhibitors are in open or partially open conformations, indicating that binding of the inhibitor to the palm subdomain indirectly restricts the conformational space sampled by the finger and thumb subdomains.

中文翻译：

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使用双电子-电子共振电子顺磁共振波谱探测HIV-1逆转录酶的手指和拇指亚域的构象状态

通过Q波段双电子-电子共振脉冲电子顺磁共振波谱研究了HIV-1逆转录酶p66亚基的手指和拇指亚域采样的构型空间，这是一种确定一氧化二氮自旋对之间长距离的方法-通过表面工程化的半胱氨酸残基引入的标记。检查了四个构建体，每个构建体在p66亚基中包含两个旋转标记，一个在手指亚结构域中，另一个在拇指亚结构域中。在未配体状态下，观察到手指和拇指亚域的打开和闭合构型，这些状态之间的分布由自旋标记和相关突变调节，这与未配位态以闭合构象结晶的晶体学数据相反。添加双链DNA后，所有构建体均采用与以前的晶体学数据一致的开放构象，在该晶体学中，拇指和手指亚结构域的位置是通过与结合的寡核苷酸双链体（DNA或DNA / RNA）接触来确定的。同样，具有五种不同的非核苷类逆转录酶抑制剂的二元复合物呈开放或部分开放构型，表明抑制剂与手掌亚结构域的结合间接限制了手指和拇指亚结构域采样的构象空间。