Veverka Václav, Bauerová Helena, Zábranský Ales, Lang Jan, Ruml Tomás, Pichová Iva, Hrabal Richard
NMR Laboratory, Institute of Chemical Technology in Prague, Technická, 5, Prague CZ-166 28, Czech Republic.
J Mol Biol. 2003 Oct 31;333(4):771-80. doi: 10.1016/j.jmb.2003.08.049.
The assembly of Mason-Pfizer monkey virus Gag polyproteins into immature capsids and their cleavage by the encoded protease are temporally and spatially separated processes, making the virus a particularly useful model for investigation of protease activation. Here we present a high resolution NMR structure of a fully folded monomer of a 12 kDa M-PMV protease (wt 12 PR) and of a Cys7Ala/Asp26Asn/Cys106Ala mutant (12 PR(D26N/C7A/C106A)). The overall structures of both wt 12 PR and 12 PR(D26N/C7A/C106A) follow the conservative structural motif of other retroviral proteases. The most prominent difference from the canonical fold of retroviral proteases is the absence of the interfacial beta-sheet, which leads to the loss of the principal force stabilizing the dimer of M-PMV PR. The monomer-dimer equilibrium can be shifted in favor of the dimer by adding a substrate or an inhibitor, partially compensating for the missing role of the beta-sheet. We also show that cysteines C7 and C106 play a crucial role in stabilizing the dimer and consequently increasing the proteolytic activity of M-PMV PR. This is consistent with the role of reversible oxidative modification of the cysteine residues in the regulation of the maturation of assembled M-PMV capsids in the cytoplasm.
梅森- Pfizer猴病毒Gag多聚蛋白组装成未成熟衣壳以及它们被编码的蛋白酶切割是在时间和空间上分离的过程，这使得该病毒成为研究蛋白酶激活的一个特别有用的模型。在此，我们展示了12 kDa的M-PMV蛋白酶（野生型12 PR）和一个Cys7Ala/Asp26Asn/Cys106Ala突变体（12 PR(D26N/C7A/C106A)）的一个完全折叠单体的高分辨率核磁共振结构。野生型12 PR和12 PR(D26N/C7A/C106A)的整体结构都遵循其他逆转录病毒蛋白酶的保守结构基序。与逆转录病毒蛋白酶的典型折叠最显著的区别是没有界面β-折叠，这导致了稳定M-PMV PR二聚体的主要力量的丧失。通过添加底物或抑制剂可以使单体-二聚体平衡向有利于二聚体的方向移动，部分补偿β-折叠缺失的作用。我们还表明，半胱氨酸C7和C106在稳定二聚体并因此增加M-PMV PR的蛋白水解活性方面起着关键作用。这与细胞质中组装的M-PMV衣壳成熟调节中半胱氨酸残基的可逆氧化修饰的作用是一致的。
