NMR solution structures of the apo and peptide-inhibited human rhinovirus 3C protease, (Serotype 14): Structural and dynamic comparison
NMR solution structures of the apo and peptide-inhibited human rhinovirus 3C protease, (Serotype 14): Structural and dynamic comparison
复制标题
DOI：
10.1021/bi7010866
复制
发表时间：
2007-11-13
期刊：
影响因子：
2.9
通讯作者：
Wishart, David S.
中科院分区：
文献类型：
--
作者：
Bjorndahl, Trent C.;Andrew, Lena C.;Wishart, David S.
人类鼻病毒（HRV）是一种阳性RNA病毒，约30%的“普通感冒”是由它引起的。它依靠182残基半胱氨酸蛋白酶（3C）对其单基因产物进行蛋白水解处理。体外和体内对这种酶的抑制一致证明了病毒复制的停止。这表明3C蛋白酶抑制剂可以作为良好的候选药物。然而，在110多种已知鼻病毒血清型中存在显著的蛋白水解底物多样性。为了研究这种变异性，我们利用核磁共振分析了鼻病毒血清型143c蛋白酶（13亚属）与肽（乙酰- lealfq -乙基丙酸）抑制剂共价结合的结构。抑制剂结合结构的总体rmsd为0.82埃（主原子）和1.49埃（所有重原子）。将与抑制剂ruprintrivir结合的A亚属血清2型HRV 3C蛋白酶（51%序列同一性）的x射线结构进行比较，可以确定涉及近端底物结合的保守分子间相互作用以及可能解释SAR研究中观察到的变异性的亚属差异。为了更好地表征3C蛋白酶，研究载脂蛋白和结合态之间的结构和动力学差异，我们还求解了载脂蛋白形式的溶液结构。载子结构在主链原子上的总体rmsd为1.07 +/- 0.17埃，比抑制酶（2130F.pdb）的rmsd大0.25埃。这种增加定位于酶的c端p桶结构域，该结构域负责识别和结合蛋白水解底物。酰胺氢交换动力学揭示了两种酶状态之间的巨大差异。此外，与抑制状态相比，在载脂蛋白状态下，许多残基表现出交换拓宽的酰胺核磁共振信号。这些残基中的大多数与蛋白水解底物相互作用有关。
The human rhinovirus (HRV) is a positive sense RNA virus responsible for about 30% of "common colds". It relies on a 182 residue cysteine protease (3C) to proteolytically process its single gene product. Inhibition of this enzyme in vitro and in vivo has consistently demonstrated cessation of viral replication. This suggests that 3C protease inhibitors could serve as good drug candidates. However, significant proteolytic substrate diversity exists within the 110+ known rhinovirus serotypes. To investigate this variability we used NMR to solve the structure of the rhinovirus serotype 14 3C protease (subgenus 13) covalently bound to a peptide (acetyl-LEALFQ-ethylpropionate) inhibitor. The inhibitor-bound structure was determined to an overall rmsd of 0.82 angstrom (backbone atoms) and 1.49 angstrom (all heavy atoms). Comparison with the X-ray structure of the serotype 2 HRV 3C protease from subgenus A (51% sequence identity) bound to the inhibitor ruprintrivir allowed the identification of conserved intermolecular interactions involved in proximal substrate binding as well as subgenus differences that might account for the variability observed in SAR studies. To better characterize the 3C protease and investigate the structural and dynamic differences between the apo and bound states we also solved the solution structure of the apo form. The apo structure has an overall rmsd of 1.07 +/- 0.17 angstrom over backbone atoms, which is greater by 0.25 angstrom than what is seen for the inhibited enzyme (2130F.pdb). This increase is localized to the enzyme's C-terminal P-barrel domain, which is responsible for recognizing and binding proteolytic substrates. Amide hydrogen exchange dynamics revealed dramatic differences between the two enzyme states. Furthermore, a number of residues exhibited exchange-broadened amide NMR signals in the apo state compared to the inhibited state. The majority of these residues are associated with proteolytic substrate interaction.