Structural modeling of RdRp NSP8-NSP12-NSP7-NSP8 multimer
We modeled the 3D structures of SARS-CoV-2 NSP7, NSP8, and NSP12 using corresponding proteins from the recently resolved SARS-CoV multimer as templates (PDB ID: 6NUR). Pairwise sequence alignment revealed sequence identities of 98.8% between NSP7, 97.5% NSP8 and 96.4% NSP12 of SARS-CoV and SARS-CoV-2 (Figure 2d). Further analyses identified regions in the SARS-CoV-2 NSP12 with constituent variations as it compares to SARS-CoV. Findings revealed high conservations at the fingers (residues 398-581, 628-687) and thumb (residues 816-919) domain which applies to the RNA/NTP binding grip. Most variations were observed across the N-terminal (residues 1-397) and palm (residues 582-627, 688-815) domains of both proteins. Variations across the NSP7 and NSP8 of SARS-CoV and SARS-CoV-2 were also indicated. More so, the N-terminal of SARS-CoV-2 NSP8 were truncated likewise the NSP7 C-terminal since they were not structurally resolved in 6NUR. 1
Structural alignment of the HADDOCK-derived complexes with ATP-bound poliovirus RdRp helped identify the ATP-bound multimer. Analyses of binding interfaces in the modeled SARS- CoV-2 NSP8-NSP12-NSP7-NSP8 multimer revealed that the NSP7-NSP8 dimer was bound at loop interfaces of the finger-thumb domain (residues 408-414, 425-447, 552) of NSP12 while regions with residues 257-275, 322-348 and 367-407 served as a binding interface for the second NSP8 subunit. These details agreed with findings from previous structural studies 1 and were used for flexible protein-protein docking.
Molecular docking yielded 10 Rem-P3 conformers among which the topmost ranked with a docking score of -7.4 kcal mol-1 was selected. This was aligned with the crystallized ATP molecule and yielded an RMSD of 0.2 Å. This further indicated that the inhibitor and substrate were properly aligned at the nucleoside pocket of SARS-CoV-2.
Remdesivir systematically disintegrates the SARS-CoV-2 RdRp assembly
To understand possible effects of Rem-P3 and ATP on the SARS-CoV-2 NSP complex, snapshots were taken from the resulting trajectories at various time-frames as the simulation proceeded. Comparative visual analysis was then carried out for the unbound, Rem-P3 - and ATP-bound systems.
We observed that the RdRp-NSP multimer remained as a unit in the unbound and ATP-bound systems from the initial to the final frames while dissimilar structural occurrences were observed in the Rem-P3 -bound complex (Figure 3).
Time-based conformational sampling revealed that the RdRp-NSP assembly was intact in the presence of Rem-P3 until around 150ns when the NSP8 subunit was firstly detached from its NSP7 counterpart into the solvent environment. This was then followed by the displacement of the NSP7 subunit from the loop interface of the NSP12-thumb domain at the next 20ns time- frame (170ns). Besides, we observed that the second NSP8 subunit was minimally displaced from its binding interface on NSP12. The disintegrated RdRp-NSP complex was maintained until the end of the simulation (Figure 4).
Based on previous studies involving SARS-CoV, NSP7 and NSP8 play important roles in de novo initiation, primer extension, RNA synthesis, and replication, particularly when complexed with NSP12. 18,33,34. Also, an assemblage of the NSP7-NSP8 heterodimer with NSP12 reportedly enhanced its RNA binding and polymerase activities. 8,18
Therefore, the ability of Rem-P3 to disrupt the RdRp-NSP assembly in SARS-CoV-2 could correlate with its inhibitory activity against the virus. This is a major finding that possibly explains its reported efficacy in the treatment of COVID-19 infections.
We further investigated the effects of both compounds on the integrity of the NSP12 multimer over the simulation period by comparing parameters for the unbound, ATP-bound and Rem-P3 - bound systems. Firstly, we estimated the stability of the complexes using the RMSD metrics and result plotted in Figure 5a. As shown, the unbound and ATP-bound complexes were stable from the beginning to the end of the simulations. On the contrary, the Rem-P3 -bound NSP complex was structurally stable (RMSD < 2Å) until ~140ns where high instability was observed, characterized by an unusual spike in Cα motions after which the system was restabilized until the end of the run. This observation could correlate with the disintegration of the NSP7-NSP8 heterodimer from the NSP12 polymerase as reported above (Figure 4) thereby explaining high conformational perturbations induced by Rem-P3 when bound to the SARS-CoV-2 RdRp-NSP complex.
A possible explanation is that the disintegration of the NSP7-NSP8 heterodimer could further destabilize the NSP12 subunit, which could, in turn, affect binding interfaces for other NSPs necessary for RNA replication 1,18,34. Another interesting and important finding was that the ATP-bound NSP-complex was more stable than the unbound system.
This could imply that the binding of ATP to NSP12 further enhanced the integrity of SARS- CoV-2 NSP8-NSP12-NSP7-NSP8 complex. To minimize entropical effects, we defined finally equilibrated RMSDs (FE-RMSDs) from the ultimate time frames (170-200ns) where the systems stabilized. As seen in Figure 5b, huge discrepancies exist between the stabilities of the simulated complexes, with a high FE-RMSD peak for the Rem-P3 bound system. This could correlate with the systematic disintegration of the RdRp-NSP complex induced by Rem-P3 relative to ATP which rather stabilized the complex.
Estimated mean FE-RMSDs of the systems were in the order ATP < Unbound < Rem-P3 as presented in Supplementary Table 1. These stable time-frames were then utilized for subsequent conformational analyses.
Also, we projected the motions of the RdRp-NSP complexes along two principal components (PC1 vs PC2). From the PCA plot in Supplementary Figure S1, we observed similar directions of motions among the unbound and ATP-bound RdRp-NSPs while a more dispersed motion pattern was observed for the Rem-P3 -bound complex.
We further monitored systemic fluctuations among constituent residues of each NSP subunits using the FE-RMSF metrics, derived from the stable time-frames. Our findings revealed that per- residual motions were relatively high in the Rem-P3 -bound NSP complex compared to ATP- bound and unbound systems (Supplementary Figure S2). Collatively, the mean FE-RMSF was lowest in the ATP-system further indicative of the importance of ATP in stabilizing the RdRp- NSP multimeric complex. Estimated mean FE-RMSFs of the systems also followed in the ATP < Unbound < Rem-P3 order.
We then mapped out the distinct subunits of the RdRp-NSP complex (including NSP12 sub- domains) and measured their fluctuations with respect to the binding of the compounds.
Our findings further emphasized the perturbative effects of Rem-P3 across the NSP8-NSP12- NSP7-NSP8 assembly. The disruptive effects of Rem-P3 binding were most pronounced in the NSP7-bound NSP8 heterodimer (FE-RMSF = 41.6Å) and could have led to their detachment from the finger loop interfaces, which was also highly perturbed (FE-RMSF = 9.0Å) according to our calculations (Figure 6). Cumulatively, high Cα motions induced by Rem-P3 at the NSP12 subdomains affected the binding and stability of associated NSPs at the interfaces (Supplementary Table 2). More so, high fluctuations induced by Rem-P3 among residues of the NSP12 N-terminal and finger subdomains could have disrupted interface interactions with the second NSP8 subunit (NSP8II), which also exhibited high structural perturbations (FE-RMSF = 11.9 Å, 9.0Å).
Interestingly, the NSP12 subdomains, in addition to other NSPs (7 and 8) exhibited minimal residual motions in the presence of ATP, indicative of the stability of the NSP complex, even compared to the unbound system. Hence, we can presume, that the binding of ATP further enhanced structural integrity across the RdRp-NSP assembly.
Differential stabilities of the NSP12-nucleoside sites were further evaluated, and as observed (Figure 5c), Rem-P3-bound nucleoside site was more unstable compared to the ATP-bound and unbound sites. This could suggest a possible correlation between the activities of Rem-P3 at the nucleoside pocket and its disruptive effects on the entire RdRp-NSP complex.
Also, Rem-P3 induced high Ca motions at the nucleoside site as further estimated using the FE- RoG metrics, which is also an indicator of structural compactness 35. Relatively, the nucleoside side was more compact when bound by ATP, even compared to the unbound NSP12-pocket (Fig 5d).
Furthermore, we projected trajectorial motions of the compounds at the active pockets with respect to their binding activities. From the PCA plot, we could observe that Rem-P3 exhibited highly unstable motions at the nucleoside pocket while ATP was coordinately bound with more compact motions indicative of its systemic stability.
Complementarily, we masked the compounds for FE-RMSD calculations to further determine their binding stability. As shown in Figure 7, Rem-P3 was highly unstable (FE-RMSD = 3.5Å ± 0.5 ) while ATP demonstrated stable motions at the binding pocket (FE-RMSD = 1.5Å ± 0.8).
Also, we observed that ATP assumed a folded conformation at the hydrophobic pocket of the NSP12 subunit, which could have accounted for the compact site architecture (Figure 7d). On the contrary, Rem-P3 exhibited an extended conformation with its cyano-adenosine ring stacked in between a visible hydrophobic patch (Figure 7b). The effect of this Rem-P3 binding mode could be seen in the pocket which was less compact.
Using average structures for the Rem-P3– and ATP-bound complexes, we closely analyzed relative orientations at the NSP12-nucleoside site and complementary interactions (Figure 8). As mentioned above, ATP ultimately assumed a compact conformation, with an inwardly folded P3 tail while Rem-P3 rather exhibited a linearly stretched conformation.
As observed, its cyano-nucleotide portion extended into the pocket created by D452, T556, V557, T680, S681, and S682 (finger domain). This ring orientation could allow for high-affinity (NH---O) interactions with D452 and T556, which could, in turn, hold the cyano-adenosine ring in place. More so, its P3 tail extends into the palm domain towards K798, D618, K621, R553, and D623. Also, the 3,4 dihydroxyoxolan ring was coordinated by N691, S759, and D760. These findings, altogether, indicate an orientation that supports its inhibitory activities. Relatively, we could observe that the P3 tail of ATP was folded away from D618 and K798, an important residue-pair responsible SARS-CoV RdRp polymerase activity as previously reported. 36
Binding free energy calculations and decomposition
The extent to which the compounds bind to the RdRp-NSP complex was determined using the MM/PBSA method. 37,38 Stable time-frames (170-200ns) were also selected to minimize entropical effects and our results are presented in Table 1. Accordingly, Rem-P3 was strongly bound than ATP with ∆Gbind difference of -13.2 kcal mol-1.
Table 1. Interaction energy profiles of Rem-P3 and ATP with RdRp-NSP12
Energy components (kcal mol-1)
|
Complexes
|
Rem-P3
|
ATP
|
∆EvdW
|
-44.4±0.3
|
-19.0±0.5
|
∆Eele
|
-38.7±0.8
|
-39.2±0.8
|
∆Ggas
|
-83.2±1.0
|
-58.2±0.9
|
∆EGB
|
54.9±0.7
|
41.1±0.4
|
∆Enp,sol
|
-5.4±0.04
|
-3.4±0.1
|
∆Gsolv
|
49.5±0.7
|
37.7±0.3
|
∆H
|
-33.7±0.6
|
-20.5±0.8
|
-T∆S
|
0.3±0.02
|
0.01±0.02
|
∆Gbind
|
-33.4±0.4
|
-20.5±0.8
|
ΔEele = electrostatic energy; ΔEvdW = van der Waals energy; ΔGbind = total binding free energy; ΔGsolv = solvation free energy ΔGgas = gas phase free energy; ΔGGB = polar desolvation energy; ΔEnp,sol non-polar solvation energy
This relatively higher affinity for the NSP12 nucleoside site further reflects its ability to competitively impede and replace the natural substrate. 25
Analysis of the binding components further revealed that the binding of Rem-P3 was more unfavorable in the polar region as evidenced by higher a ∆Gnp,sol value. This could suggest that Rem-P3 does not only block ATP binding but binds deeply in the hydrophobic nucleoside pocket of the NSP12 subunit.
This could favor its retention and involvement with more residues of the pocket. Also, vdW contributions to Rem-P3 binding was relatively higher, which may compensate electrostatic (∆Eele) effects that were negated by unfavorable polar solvation energies (∆Gsolv). Cumulatively, ∆EvdW and ∆Eele highly favored gas-phase interactions (∆Ggas) of Rem-P3 relative to ATP.
Also, we calculated the energies of individual residues and their respective contributions to the disparate binding of both compounds. Energy contributions > -1 kcal mol-1 were considered favorable and results are presented in Figure 9.
As shown, total energy contributions to Rem-P3 were highly favorable for D452 (-4.1 kcal mol- 1), D760 (-3.6 kcal mol-1), S682 (-2.8 kcal mol-1), R555 (-2.0 kcal mol-1), T556 (-1.6 kcal mol-1), T687 (-1.1 kcal mol-1), V557 (-1.0 kcal mol-1) and T680 (-1.1 kcal mol-1) in that order.
This could further emphasize the importance of D452 in enhancing Rem-P3 binding via interaction with its cyano-adenosine portion, and D760 via interactions with its 3,4 dihydroxyoxolan ring (Figure 8a). Relative to ATP, most of these energies were reduced and highly unfavorable. For instance, D425 and R553 contributed unfavorable energies (+ve) of 1.0 and 5.3 kcal mol-1 to ATP which could minimize its interaction affinity compared to Rem-P3. However, S549 (-1.1 kcal mol-1), R555 (-5.9 kcal mol-1), K621 (-2.5 kcal mol-1) and D623 (-2.5 kcal mol-1) all contributed favorably to ATP binding.
The roles of D618 and K798 to the binding of Rem-P3 were further defined with electrostatic contributions of -11.4 and -1.2 kcal mol-1 respectively. On the contrary, electrostatic effects by D618 were highly unfavorable for ATP (10.1 kcal mol-1).
This could suggest that ATP does not interfere with the roles mediated by D618 during nucleotide polymerization. 36 More so, while D452 electrostatically favored Rem-P3 binding (-5.3 kcal mol-1), an unfavorable contribution of +12.5 kcal mol-1 was estimated for ATP. Similar Rem-P3 → ATP (∆Eele) transitions were observed for T556 (-5.4 → 1.9 kcal mol-1), D623 (-10.1 → 9.0 kcal mol-1), D760 (-11.2 → 9.2 kcal mol-1) and D761 (-8.3 → 7.4 kcal mol-1). Presumably, residues that favored Rem-P3 binding at the nucleotide site of RdRp-NSP12 rather contributed unfavorably to ATP binding, which could limit ATP affinity in the region. Also, in Rem-P3, unfavorable ∆Eele contributions were highly compensated for by per-residue ∆EvdW energies which were favorable in R555 (-2.9 kcal mol-1), T556 (-1.4 kcal mol-1), V557 (1.0 kcal mol-1), D623 (-2.3 kcal mol-1), R624 (-1.4 kcal mol-1), T680 (-1.1 kcal mol-1), S681 (-1.4 kcal mol-1) and S682 (-2.8 kcal mol-1). These were decreased to -1.7 kcal mol-1 (R555), -0.4 kcal mol- 1 (T556), +0.4 kcal mol-1 (D623) and -0.7 kcal mol-1 (R624) in the ATP-complex.
Taken together, we could presume that Rem-P3 binds uncoordinatedly at the NSP12 nucleoside site, interacting with a high number of residues along its path. This binding pattern favorably enhanced its affinity and, perhaps, longer retention at the NSP12 site until it exerts maximum destabilizing effects sufficient to disrupt the RdRp-NSP multimeric assembly.
Relatively, the ability of ATP to fold its flexible P3 tail away from D618 and K798 in a compact conformation indicates a selective and coordinated binding, favorable for the nucleotide polymerization process.
In this study, we investigated the differential binding of Rem-P3 and ATP to the RdRp-NSP assembly. This involved subunit binding of an NSP7-NSP8 heterodimer to NSP12 while a second NSP8 (NSP8II) was bound at a distant site. The complex, although not currently available for SARS-CoV-2 at the time of filing this report, was modeled using a structurally similar SARS-CoV RdRp-NSP complex (PDB ID: 6NUR). The Rem-P3-, ATP- and Unbound complexes were subjected to GPU-accelerated molecular dynamics simulation of 600ns after which the trajectories were sampled conformationally. Findings revealed that the RdRp-NSP complex was more stable in the presence of ATP, even compared to the unbound system while on the other hand, Rem-P3 appeared to disintegrate the complex. The NSP7-NSP8 heterodimer was firstly detached at ~150ns from the NSP12 while the second NSP8 (NSP8II) subunit was seen to be minimally displaced at the ultimate time-frame. Conformational analysis performed with RMSD and RoG parameters further revealed that Rem-P3 induced a high degree of structural instability compared to the ATP and unbound system. RMSF analysis revealed relatively higher subdomain perturbations in the Rem-P3-bound RdRp-NSP complex. Also, analysis of ligand motions revealed that ATP exhibited a more stable and compact motion compared to Rem-P3, which could have also reflected on the active site, which was less deviated and more compact in the ATP-system. Analysis of binding dynamics revealed that Rem-P3 exhibited an extended conformation that allowed it to interact with D542, T556, V557, T680, S681 and S682 of the NSP12-finger domain via its cyano-adenosine ring while its 3,4 dihydroxyoxolan ring oriented towards D760 and N961 forming high-affinity H-bonds. Also, its P3 tail was extended towards the palm domain, interacting with D618 and K798. This binding pattern clearly interferes with the roles mediated by D618 and K798 in RNA replication process. On the contrary, ATP exhibited a more selective and coordinated binding wherein it assumed a folded (compact) conformation away from these residues. Binding energy value of -33.4 kcal mol-1 was estimated for Rem-P3 while ATP had a ΔGbind estimate of -20.5 kcal mol-1. This further reflected the ability of Rem-P3 to competitively replace ATP at the NSP12-nucleoside pocket. Per-residue energy decomposition further emphasized the roles of D542, T556, V557, T680, S681, S682, D618 and K798 to the high-affinity binding of Rem-P3. We believe findings from this study will significantly contribute to drug design endeavors, particularly structure- based approaches. This could yield novel compounds that can bind selectively and strongly to the NSP12-nucleoside site and disrupt interactions with other non-structural proteins.