Molecular mechanism for conformational activation of SARS-CoV-2 RNA polymerase by nucleoside triphosphate

Abstract

It has been shown that the nucleotide analogs such as remdesivir and molnupiravir are promising drug candidates to treat COVID-19 infection, by targeting SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). Characterizing the molecular mechanism of RNA replication by RdRp is crucial to understand how these inhibitors work. Here we carried out molecular dynamics simulations coupled with enhanced sampling techniques to study the substrate binding mechanism and conformational dynamics of the SARS-CoV-2 RdRp complex based on recently available cryo-EM structures. Our results reveal the transition pathways for active site closure, which is required for substrate catalysis. Structural bioinformatics analysis indicates such motions exist in other homologous viral polymerases. Furthermore, we demonstrate that the RNA-binding cleft motion is coupled to the coordination of two magnesium ions at the active site. Motif G is found to play a key role in nucleotide positioning during the activation process. By applying the Gaussian accelerated molecular dynamics (GaMD) to enhance the sampling of the substrate dynamics, we find a key intermediate state that precedes the tight binding of the nucleoside triphosphate. The simulations provide metastable RdRp conformations at the active site, which can help the drug discovery using various approved nucleotide inhibitors. It has been shown that the nucleotide analogs such as remdesivir and molnupiravir are promising drug candidates to treat COVID-19 infection, by targeting SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). Characterizing the molecular mechanism of RNA replication by RdRp is crucial to understand how these inhibitors work. Here we carried out molecular dynamics simulations coupled with enhanced sampling techniques to study the substrate binding mechanism and conformational dynamics of the SARS-CoV-2 RdRp complex based on recently available cryo-EM structures. Our results reveal the transition pathways for active site closure, which is required for substrate catalysis. Structural bioinformatics analysis indicates such motions exist in other homologous viral polymerases. Furthermore, we demonstrate that the RNA-binding cleft motion is coupled to the coordination of two magnesium ions at the active site. Motif G is found to play a key role in nucleotide positioning during the activation process. By applying the Gaussian accelerated molecular dynamics (GaMD) to enhance the sampling of the substrate dynamics, we find a key intermediate state that precedes the tight binding of the nucleoside triphosphate. The simulations provide metastable RdRp conformations at the active site, which can help the drug discovery using various approved nucleotide inhibitors.