The investigation of the inhibition effects of substituting Mg ²⁺ with Ca ²⁺ and Zn ²⁺ on SARS-CoV-2 RNA-dependent RNA polymerase

Metal ions are essential for the catalytic function of RNA-dependent RNA polymerase (RdRp) from many coronaviruses. However, different metal ions exert distinct effects on their catalytic efficiency and the underlying molecular mechanisms remain poorly understood. In this study, various computational approaches were employed to investigate the potential for substituting Mg²⁺ with various metal ions in the SARS-CoV-2 RdRp active site and to evaluate their impact on its activity. Predictions from the Metal Ion-Binding site prediction and docking (MIB) server indicate that Ca²⁺, Zn²⁺, Mn²⁺, and Co²⁺ possess the potential to substitute for Mg²⁺ within the SARS-CoV-2 RdRp active site. However, results from molecular docking and molecular dynamics (MD) simulations indicate that Ca²⁺ and Zn²⁺ can hinder the formation of the phosphodiester bond. Based on our structural analyses, an inhibitory mechanism was proposed as follows: (I) the nucleoside triphosphate (NTP) is slightly shifted from its optimal position due to the longer ionic radii of Ca²⁺ and Zn²⁺; (II) the NTP-RNA distance exceeds the optimal range of 3.5-4 Å required for efficient phosphodiester bond formation; (III) the critical hydrogen bond between the NTP γ-phosphate and the conserved K798 from Motif D is disrupted, leading to increasing NTP's triphosphate flexibility; and (IV) the electrostatic interactions formed between the flexible NTP triphosphate and K551 and R553 from Motif F act as a switch to prevent the NTP channel from reaching the closed-state conformation. This metal ion-dependent catalytic mechanism may be further validated through a series of <i>in vitro</i> and <i>in vivo</i> approaches.