Louis J M, Clore G M, Gronenborn A M
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
Nat Struct Biol. 1999 Sep;6(9):868-75. doi: 10.1038/12327.
In the Gag-Pol polyprotein of HIV-1, the 99-amino acid protease is flanked at its N-terminus by a transframe region (TFR) composed of the transframe octapeptide (TFP) and 48 amino acids of the p6pol, separated by a protease cleavage site. The intact precursor (TFP-p6pol-PR) has very low dimer stability relative to that of the mature enzyme and exhibits negligible levels of stable tertiary structure. Thus, the TFR functions by destabilizing the native structure, unlike proregions found in zymogen forms of monomeric aspartic proteases. Cleavage at the p6pol-PR site to release a free N-terminus of protease is concomitant with the appearance of enzymatic activity and formation of a stable tertiary structure that is characteristic of the mature protease as demonstrated by nuclear magnetic resonance. The release of the mature protease from the precursor can either occur in two steps at pH values of 4 to 6 or in a single step above pH 6. The mature protease forms a dimer through a four-stranded beta-sheet at the interface. Residues 1-4 of the mature protease from each subunit constitute the outer strands of the beta-sheet, and are essential for maintaining the stability of the free protease but are not a prerequisite for the formation of tertiary structure and catalytic activity. Our experimental results provide the basis for the model proposed here for the regulation of the HIV-1 protease in the viral replication cycle.
在HIV-1的Gag-Pol多聚蛋白中，由99个氨基酸组成的蛋白酶在其N端侧翼是一个移码区（TFR），该移码区由移码八肽（TFP）和p6pol的48个氨基酸组成，两者被一个蛋白酶切割位点隔开。完整的前体（TFP-p6pol-PR）相对于成熟酶而言，其二聚体稳定性非常低，并且稳定的三级结构水平可忽略不计。因此，与单体天冬氨酸蛋白酶的酶原形式中发现的前区不同，TFR通过破坏天然结构起作用。在p6pol-PR位点的切割以释放蛋白酶的游离N端，这与酶活性的出现以及成熟蛋白酶特有的稳定三级结构的形成同时发生，核磁共振已证实了这一点。成熟蛋白酶从前体的释放可以在pH值为4至6时分两步进行，或者在pH值高于6时一步完成。成熟蛋白酶通过界面处的四链β折叠形成二聚体。每个亚基的成熟蛋白酶的第1至4位残基构成β折叠的外侧链，对于维持游离蛋白酶的稳定性至关重要，但不是三级结构形成和催化活性的先决条件。我们的实验结果为此处提出的HIV-1蛋白酶在病毒复制周期中的调节模型提供了依据。
