Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai Jiao Tong University, School of Medicine, Shanghai 20025, China.
Department of VIP Clinic, Changhai Hospital, Navy Military Medical University, Shanghai, 200433, China.
Phys Chem Chem Phys. 2021 Jun 2;23(21):12204-12215. doi: 10.1039/d0cp06360f.
Hepatitis C virus (HCV) is a notorious member of the Flaviviridae family of enveloped, positive-strand RNA viruses. Non-structural protein 5A (NS5A) plays a key role in HCV replication and assembly. NS5A is a multi-domain protein which includes an N-terminal amphipathic membrane anchoring alpha helix, a highly structured domain-1, and two intrinsically disordered domains 2-3. The highly structured domain-1 contains a zinc finger (Zf)-site, and binding of zinc stabilizes the overall structure, while ejection of this zinc from the Zf-site destabilizes the overall structure. Therefore, NS5A is an attractive target for anti-HCV therapy by disulfiram, through ejection of zinc from the Zf-site. However, the zinc ejection mechanism is poorly understood. To disclose this mechanism based on three different states, A-state (NS5A protein), B-state (NS5A + Zn), and C-state (NS5A + Zn + disulfiram), we have performed molecular dynamics (MD) simulation in tandem with DFT calculations in the current study. The MD results indicate that disulfiram triggers Zn ejection from the Zf-site predominantly through altering the overall conformation ensemble. On the other hand, the DFT assessment demonstrates that the Zn adopts a tetrahedral configuration at the Zf-site with four Cys residues, which indicates a stable protein structure morphology. Disulfiram binding induces major conformational changes at the Zf-site, introduces new interactions of Cys39 with disulfiram, and further weakens the interaction of this residue with Zn, causing ejection of zinc from the Zf-site. The proposed mechanism elucidates the therapeutic potential of disulfiram and offers theoretical guidance for the advancement of drug candidates.
丙型肝炎病毒 (HCV) 是黄病毒科有包膜、正链 RNA 病毒的臭名昭著的成员。非结构蛋白 5A (NS5A) 在 HCV 复制和组装中发挥关键作用。NS5A 是一种多结构域蛋白，包括一个 N 端两亲性膜锚定α螺旋、一个高度结构化的结构域 1 和两个固有无序结构域 2-3。高度结构化的结构域 1 包含一个锌指 (Zf)-位点，锌的结合稳定了整体结构，而从 Zf-位点排出锌则会使整体结构不稳定。因此，NS5A 是通过从 Zf-位点排出锌来抑制 HCV 的治疗的有吸引力的靶点。然而，锌排出机制知之甚少。为了基于三种不同状态（A 态（NS5A 蛋白）、B 态（NS5A+Zn）和 C 态（NS5A+Zn+disulfiram））揭示这一机制，我们在当前研究中结合 DFT 计算进行了分子动力学 (MD) 模拟。MD 结果表明，disulfiram 主要通过改变整体构象集触发 Zn 从 Zf-位点排出。另一方面，DFT 评估表明 Zn 在 Zf-位点采用四面体构型与四个 Cys 残基结合，这表明蛋白质结构形态稳定。disulfiram 结合诱导 Zf-位点的主要构象变化，引入 Cys39 与 disulfiram 的新相互作用，并进一步削弱该残基与 Zn 的相互作用，导致锌从 Zf-位点排出。所提出的机制阐明了 disulfiram 的治疗潜力，并为药物候选物的发展提供了理论指导。
