Using molecular dynamics simulations, we describe how crowded environments affect the internal dynamics and diffusion of the hepatitis C virus proteases NS3/4A. This protease plays a key role in viral replication and is successfully used as a target for antiviral treatment. The NS3 enzyme requires a peptide cofactor, called NS4A, with its central part interacting with the NS3 β-sheet, and flexible, protruding terminal tails that are unstructured in water solution. The simulations describe the enzyme and water molecules at atomistic resolution, whereas crowders are modeled via either all-atom or coarse-grained models to emphasize different aspects of crowding. Crowders reflect the polyethylene glycol (PEG) molecules used in the experiments to mimic the crowded surrounding. A bead-shell model of folded coarse-grained PEG molecules considers mainly the excluded volume effect, whereas all-atom PEG models afford more protein-like crowder interactions. Circular dichroism spectroscopy experiments of the NS4A N-terminal tail show that a helical structure is formed in the presence of PEG crowders. The simulations suggest that crowding may assist in the formation of an NS4A helical fragment, positioned exactly where a transmembrane helix would fold upon the NS4A contact with the membrane. In addition, partially interactive PEGs help the NS4A N-tail to detach from the protease surface, thus enabling the process of helix insertion and potentially helping the virus establish a replication machinery needed to produce new viruses. Results point to an active role of crowding in assisting structural changes in disordered protein fragments that are necessary for their biological function.

使用分子动力学模拟，我们描述了拥挤的环境如何影响丙型肝炎病毒蛋白酶 NS3/4A 的内部动力学和扩散。这种蛋白酶在病毒复制中起着关键作用，并成功地用作抗病毒治疗的靶标。NS3 酶需要一种称为 NS4A 的肽辅助因子，其中心部分与 NS3 β相互作用-片状、柔性、突出的末端尾巴，在水溶液中是非结构化的。模拟以原子分辨率描述酶和水分子，而拥挤者则通过全原子或粗粒度模型建模，以强调拥挤的不同方面。拥挤反映了实验中用于模拟拥挤环境的聚乙二醇 (PEG) 分子。折叠粗粒度 PEG 分子的珠壳模型主要考虑排除的体积效应，而全原子 PEG 模型提供更多类似蛋白质的拥挤相互作用。NS4A N 末端尾部的圆二色光谱实验表明，在 PEG 拥挤的情况下形成螺旋结构。模拟表明拥挤可能有助于 NS4A 螺旋片段的形成，准确定位在跨膜螺旋在 NS4A 与膜接触时折叠的位置。此外，部分相互作用的 PEG 有助于 NS4A N 尾从蛋白酶表面分离，从而实现螺旋插入过程，并可能帮助病毒建立产生新病毒所需的复制机制。结果表明，拥挤在协助实现其生物学功能所必需的无序蛋白质片段的结构变化方面发挥着积极作用。