University of Lille, CNRS, UMR 8576, UGSF-Unité de Glycobiologie Structurale et Fonctionnelle, F-59000 Lille, France.
Department of Infectious Diseases, Molecular Virology, University of Heidelberg, 69120 Heidelberg, Germany.
J Biol Chem. 2019 Aug 30;294(35):13171-13185. doi: 10.1074/jbc.RA119.009537. Epub 2019 Jul 17.
Implicated in numerous human diseases, intrinsically disordered proteins (IDPs) are dynamic ensembles of interconverting conformers that often contain many proline residues. Whether and how proline conformation regulates the functional aspects of IDPs remains an open question, however. Here, we studied the disordered domain 2 of nonstructural protein 5A (NS5A-D2) of hepatitis C virus (HCV). NS5A-D2 comprises a short structural motif (PW-turn) embedded in a proline-rich sequence, whose interaction with the human prolyl isomerase cyclophilin A (CypA) is essential for viral RNA replication. Using NMR, we show here that the PW-turn motif exists in a conformational equilibrium between folded and disordered states. We found that the fraction of conformers in the NS5A-D2 ensemble that adopt the structured motif is allosterically modulated both by the / isomerization of the surrounding prolines that are CypA substrates and by substitutions conferring resistance to cyclophilin inhibitor. Moreover, we noted that this fraction is directly correlated with HCV RNA replication efficiency. We conclude that CypA can fine-tune the dynamic ensemble of the disordered NS5A-D2, thereby regulating viral RNA replication efficiency.
涉及多种人类疾病的无规卷曲蛋白质（IDPs）是动态的构象转换混合物，通常含有许多脯氨酸残基。然而，脯氨酸构象是否以及如何调节 IDPs 的功能方面仍然是一个悬而未决的问题。在这里，我们研究了丙型肝炎病毒（HCV）非结构蛋白 5A（NS5A-D2）的无规结构域 2。NS5A-D2 包含一个短的结构基序（PW-转角）嵌入在脯氨酸丰富的序列中，其与人类脯氨酰基异构酶 cyclophilin A（CypA）的相互作用对于病毒 RNA 复制是必不可少的。在这里，我们使用 NMR 表明 PW-转角基序在折叠和无规状态之间存在构象平衡。我们发现，无规卷曲 NS5A-D2 混合物中采用结构化基序的构象分数，通过周围 CypA 底物脯氨酸的 / 异构化以及赋予对环孢素抑制剂抗性的取代来进行变构调节。此外，我们注意到，该分数与 HCV RNA 复制效率直接相关。我们得出结论，CypA 可以微调无规卷曲 NS5A-D2 的动态混合物，从而调节病毒 RNA 复制效率。
