Divita G, Rittinger K, Geourjon C, Deléage G, Goody R S
Max-Planck-Institut für Medizinische Forschung, Abteilung Biophysik, Heidelberg, Germany.
J Mol Biol. 1995 Feb 3;245(5):508-21. doi: 10.1006/jmbi.1994.0042.
The dimerization processes of the human immunodeficiency virus (HIV) types 1 and 2 reverse transcriptase (RTs) from their subunits have been investigated using a number of complementary approaches (fluorescence spectroscopy, size exclusion-HPLC and polymerase activity assay). The formation of the native heterodimeric form of HIV-1 and HIV-2 RT occurs in a two step process. The first step is a concentration-dependent association of the two subunits (p66 and p51) to give a heterodimeric intermediate, which slowly isomerizes to the "mature" heterodimeric form of the enzyme. For both RTs, the first step behaves as a second order reaction with similar association rate constants (in the range of 2 x 10(4) to 4 x 10(4) M-1 s-1). This initial dimerization results in a 25% quenching of the intrinsic fluorescence and a 30% decrease in the accessibility of the tryptophan hydrophobic cluster to solvent as revealed by iodide quenching experiments and by monitoring the binding of 1-anilino-8-naphthalenesulphonate. The formation of the intermediate-RT form appears to involve hydrophobic regions of the subunits containing tryptophan residues. This intermediate form is devoid of polymerase activity, but is able to bind primer/template with high affinity. The final stage of the mature RT-heterodimer formation occurs in a slow first order reaction, which is 12-fold faster for HIV-2 (1.2 h-1) than HIV-1 RT (0.1 h-1). At micromolar concentrations, this slow isomerization constitutes the rate limiting step of the RT maturation and the structural change involved appears to be partly associated with the catalytic site, as shown using fluorescent labelled primer/template. On the basis of both the presently available X-ray structure of the HIV-1 RT and the predicted structure of HIV-2 RT, the thumb subdomain of the p51 subunit seems to be involved in this maturation step, which is probably the interaction of this domain with the RNAse H domain of the large subunit. The placement of the fingers subdomain of p51 in the palm subdomain of the p66 subunit may also be associated with formation of mature heterodimeric RTs.
已使用多种互补方法（荧光光谱法、尺寸排阻高效液相色谱法和聚合酶活性测定法）研究了人类免疫缺陷病毒1型和2型逆转录酶（RT）从其亚基形成二聚体的过程。HIV-1和HIV-2 RT天然异源二聚体形式的形成过程分两步进行。第一步是两个亚基（p66和p51）的浓度依赖性缔合，形成异源二聚体中间体，该中间体缓慢异构化为酶的“成熟”异源二聚体形式。对于两种RT，第一步均表现为二级反应，缔合速率常数相似（在2×10⁴至4×10⁴ M⁻¹ s⁻¹范围内）。如碘化物猝灭实验以及通过监测1-苯胺基-8-萘磺酸盐的结合所揭示的，这种初始二聚化导致内在荧光猝灭25%，色氨酸疏水簇对溶剂的可及性降低30%。中间体RT形式的形成似乎涉及含有色氨酸残基的亚基疏水区域。这种中间体形式没有聚合酶活性，但能够以高亲和力结合引物/模板。成熟RT异源二聚体形成的最后阶段以缓慢的一级反应发生，HIV-2（1.2 h⁻¹）比HIV-1 RT（0.1 h⁻¹）快12倍。在微摩尔浓度下，这种缓慢的异构化构成了RT成熟的限速步骤，并且所涉及的结构变化似乎部分与催化位点相关，如使用荧光标记的引物/模板所示。基于目前可得的HIV-1 RT的X射线结构和HIV-2 RT的预测结构，p51亚基的拇指结构域似乎参与了这一成熟步骤，这可能是该结构域与大亚基的核糖核酸酶H结构域的相互作用。p51的手指结构域在p66亚基的手掌结构域中的位置也可能与成熟异源二聚体RT的形成有关。
