Binding of SARS-CoV-1/2 NSP1 to DNA Polymerase α-Primase Inhibits DNA Replication through Reduction of Interaction between DNA and DNA Polymerase α-Primase

Abstract

A comprehensive understanding of the atomic level mechanism governing the binding nonstructural protein 1 of SARS-CoV-1 (SARS-CoV-1 NSP1) and SARS-CoV-2 (SARS-CoV-2 NSP1) to Pol α-primase is important to advance the development of small molecule inhibitors for the treatment COVID-19. In this study, we use both all-atom steered molecular dynamics (all-atom SMD) and coarse-grained umbrella sampling (coarse-grained US) simulations to assess the binding affinity of SARS-CoV-1 NSP1 and SARS-CoV-2 NSP1 to Pol α-primase. Our all-atom SMD and coarse-grained US simulations consistently indicate that SARS-CoV-2 NSP1 exhibits stronger affinity for Pol α-primase compared to SARS-CoV-1 NSP1, implying that SARS-CoV-2 poses a greater risk than SARS-CoV-1 in impeding DNA replication for DNA synthesis. Through an energetic decomposition analysis of the interaction energies within these complexes, we identify electrostatic interactions as the primary contributors to the observed difference in binding affinity. We found that hydrogen bonds between Asp33 and Arg616 in SARS1 NSP1-Pol α-primase, and Asp33 with Arg616 and Lys655 in SARS2 NSP1-Pol α-primase, are critical for the interaction of both SARS-CoV-1 NSP1 and SARS-CoV-2 NSP1 with Pol α-primase. Asp33 in SARS-CoV-2 NSP1 shows increased solubility and stability compared to SARS-CoV-1 NSP1, enhancing its association with Pol α-primase. This finding lays the groundwork for innovative strategies aimed at inhibiting the interaction between these entities, offering promising avenues for therapeutic intervention against COVID-19. We also estimated the binding free energy of DNA to Pol α-primase, SARS1 NSP1-Pol α-primase, and SARS2 NSP1-Pol α-primase using the MM-PBSA method. The results show the order: Pol α-primase-DNA < SARS1 NSP1-Pol α-primase-DNA < SARS2 NSP1-Pol α-primase-DNA, indicating that both SARS-CoV-1 NSP1 and SARS-CoV-2 NSP1 reduce DNA binding to Pol α-primase, suggesting impaired DNA synthesis.

1

(A) Scheme of (left) beyond its recognized function in chromosomal DNA replication during the S-phase, Pol α-primase has been hypothesized to play a cytoplasmic role in synthesizing RNA/DNA duplexes, potentially influencing the interferon response in the context of viral infections; and (right) the hypothetical mechanisms underlying the NSP1-dependent interference of SARS-CoV-2 with cytoplasmic Pol α-primase. (B) 3D structure of SARS-CoV-1 NSP1 (green-cyan) or SARS-CoV-2 NSP1 (magenta) binds to Pol α-primase, including polymerase α (blue), B subunit (yellow), Pri1 (orange), and Pri2 (green).

2

(A) Time dependence of pulling force, (B) displacement dependence of pulling force, (C) time dependence of nonequilibrium work, and (D) time dependence of nonequilibrium energy profiles of the SARS1 NSP1-Pol α-primase and SARS2 NSP1-Pol α-primase complexes. The results were averaged from 5 independent all-atom SMD runs.

3

Time dependence of (A) vdW interaction energy, (B) electrostatic interaction energy, (C) total nonbonded interaction energy, and (D) total nonbonded interaction energy of per-residue at the binding regions for a [0–t _max] time window of SARS1 NSP1-Pol α-primase (black) and SARS2 NSP1-Pol α-primase (red). The results were averaged from 5 independent all-atom SMD runs.

4

Hydrogen bond networks of (A) SARS1 NSP1-Pol α-primase and (B) SARS2 NSP1-Pol α-primase for 5 trajectories of all-atom SMD simulations for before and after F _max. Snapshots were taken at a distance of 0.1 nm before and 0.15 nm after F _max, based on the displacement-time profile. These results generated by the LigPlot.

5

1D-PMF for a [50–1000 ns] time window as a function of the RC. The left and right snapshots refer to the bound and unbound state of SARS2 NSP1-Pol α-primase. The arrow indicates the position of the cutoff distance between bound and unbound states. These results were estimated from coarse-grained US simulations using the MARTINI model.

6

(A) 3D structure of SARS-CoV-1 NSP1 or SARS-CoV-2 NSP1 binds to Pol α-primase with DNA. (B) Total nonbonded interaction energy of per-residue at the binding regions of SARS1 NSP1-Pol α-primase-DNA (black) and SARS2 NSP1-Pol α-primase-DNA (red). The results were averaged from the last 130 ns of all-atom conventional MD runs.