Mutations in several phosphorylation sites within the phosphorylation rich domain of SARS-COV-2 Nucleocapsid protein (NCp), including serines 186, 197 and 202, and adjacent arginine 203 and glycine 204 have been described and have been proposed to prevent the binding and sequestration of NCp by Protein 14-3-3. Structure modeling and thermodynamic calculation show that mutations of phosphorylation sites, phospho-serines 186, 197 and 202 to phenylalanine, leucine and asparagine, and phosphorylation recognition sites, arginine/glycine 203/204 to lysine/arginine or lysine/threonine resulted in significant destabilization of the NCp-14-3-3 complex by causing a decrease in Stability Energy (ΔG_{stability energy}) and Binding Energy (ΔΔG_{binding energy}). These results evidenced that mutations in NCp underlie a mechanism to bypass sequestration by Protein 14-3-3 which would result in enhanced dimerization of NCp, and replication, transcription and packaging of the SARS-COV-2 genome.

Introduction

SARS-COV-2 Nucleocapsid protein (NCp) plays an essential role as a co-factor in the initiation and control of the replication, transcription and packaging of the SARS-COV-2 genome [1-6]. Dimerization of SARS-COV-2 Nucleocapsid protein (NCp) is a prerequisite step in the replication, transcription and packaging of the SARS-COV-2 genome. Oligomerization of NCp is also necessary for the packaging of SARS-COV-2 genome [7-12]. Phosphorylation of NCp has been proposed to play an important role in the control of NCp functions [13-17]. A cellular response mechanism for preventing dimerization of NCp involving phosphorylation dependent sequestration of monomeric NCp by Protein 14-3-3 has been proposed [13]. Inhibiting the dimerization and oligomerization of NCp is an attractive way to control SARS-COV-2 viability, infection and virulence [13,18].

Phosphorylation of serines 197 and 206 of NCp by C-TAK-1 has been proposed to constitute the formation of the binding and sequestration sites (RNpSTP and RGTpSP) that are located in the linker region of NCp for Protein 14-3-3 [13,18]. Other phosphorylations sites, including serines 186 and 202, and threonines 198 and 205 are also present in the phosphorylation rich domain of NCp [13,18]. Serines 186, 197 and 202 become mutated to phenylalanine, leucine and asparagine in SARS-COV-2 strains/sub-strains from Iran, Spain and India respectively. Arginine 203 and glycine 204 are mutated to lysine and arginine or lysine and threonine in SARS-COV-2 strains/sub-strains from Israel, Italy, Poland, Bangladesh, Greece and Czech Republic [13]. The significance of these mutations is not known. Here, we show by computational structure model analysis, and thermodynamic calculation that mutations of the phosphorylation sites, phospho-serines 186, 197 and 202, and the adjacent amino acids, arginine 203 and glycine 204 caused decreases in the Stability Energy (ΔG_{stability energy}) and Binding Energy (ΔΔG_{binding energy}) of the NCp-Protein 14-3-3 complex. It is submitted that cells infected with SARS-COV-2 possess a cellular response mechanism for the binding and sequestration of NCp by Protein 14-3-3 involving multi-sites phosphorylation by a variety of cellular protein kinases. In counterpart, SARS-COV-2 has evolved to evade the cellular response mechanism through mutations of at least 3 phosphorylation sites, serines 186, 197 and 202 and adjacent phosphorylation recognition sites (RNpSTP and RGTpSP), arginine 203 and glycine204.

Methods

The de novo rendering of the structure of dephospho-NCp using the Quark Program pursuant to et al. [19,20] was as described previously [13,18]. Phosphorylation of SARS-COV-2 Nucleocapsid protein (NCp) was performed with FoldX using the Build Model Program pursuant to Guerois et al. and Schymkowitz, et al. [21,22]. Mutation of amino acid residues within NCp was also performed with FoldX using the Build Model Program pursuant Guerois, et al. and Schymkowitz et al. [21,22]. Docking experiments to identify the binding and sequestration of NCp by Protein 14-3-3 and dimerization of NCp were performed using the ZDOCK program pursuant to Pierce, et al. [23]. NCps rendered in this work and Protein 14-3-3 (1YZ5) based on the structure determination of Benzinger, et al. [24] were analyzed and visualized by the CCP4 Molecular Graphics Program Version 2.10.11 as described by Mc Nicolas, et al [25]. Realistic rendering of the structure of NCps were performed with the ZMM Molecular Modeling Program as described by Garden and Zhorov [26].

Determination and calculation of Stability Energy (ΔG_{stability energy}) of the protein complexes and non-complexed proteins was performed using the Stability Program of FoldX as described by Guerois et al. and Schymkowitz, et al. [21,22]. Binding Energy (ΔΔG_{binding energy}) of protein complex was determined and calculated using the Analyze Complex Program of FoldX as described by Guerois et al. and Schymkowitz, et al. [21,22]. Binding Energy Difference (ΔΔΔG_{binding energy} difference) between phospho-NCp-Protein 14-3-3 complex and phospho-NCp mutants-Protein 14-3-3 complex was calculated pursuant to Teng et al. and Nishi, et al. [27-29] from the equation: ΔΔΔG_{binding energy difference} = ΔΔG_{binding energy} (binding energy of phospho-NCp-14-3-3 complex) - ΔΔG_{binding energy} (binding energy of phospho-NCp mutants-14-3-3 complex.

Results

The consequences of mutations in the phosphorylation sites of NCp were determined with respect to binding and sequestration of NCp by Protein 14-3-3. Figures 1 and 2 summarize the dockings of phospho-serine 186-NCp and phenylalanine 186-NCp mutant with Protein 14-3-3. Both phospho-serine 186-NCp and phenylalanine 186-NCp mutant form complexes with Protein 14-3-3 with very subtle conformational change. However, the calculated Stability Energy (ΔG_{stability energy}) and Binding Energy (ΔΔG_{binding energy}) of phospho-serine 186-NCp-Protein 14-3-3 were ~605 Kcal/mol and ~215 Kcal/mol respectively while the calculated Stability Energy (ΔG_{stability energy}) and Binding Energy (ΔΔG) of phenylalanine 186-NCp mutant-Protein 14-3-3 complex was ~597 Kcal/mol and ~208 respectively. These results suggest that mutation of serine 186 of NCp to phenylalanine was accompanied by destabilization of the NCp-Protein 14-3-3 complex and reduction of the binding affinity between NCp and protein 14-3-3. The Binding Energy Difference (ΔΔΔG_{binding energy difference}) between phospho-serine 186 NCp-Protein 14-3-3 complex and phenylalanine 186-NCp mutant-Protein 14-3-3 complex was calculated to be ~ -7 Kcal/mol. Pursuant to Teng et al. and Nishi, et al. [27-29], negative Binding Energy Difference is thermodynamic evidence of destabilization and lowering of binding efficiency.

Figure 1: A: Ribbon structure (red) of phospho-serine 186-SARS-COV-2 Nucleocapsid protein (NCp) and protein 14-3-3 complex, rendered as described in method section. Phosphorylation rich domain is shown as blue spheres. B: Realistic rendering of phospho-serine 186-SARS-COV-2 Nucleocapsid protein (NCp) (yellow) and protein 14-3-3 pre-complex (red and blue).

Figure 2: A: Ribbon structure (red) of S186F-SARS-COV-2 Nucleocapsid protein (NCp) and protein 14-3-3 complex, rendered as described in method section. Blue spheres show the phosphorylation rich domain. B: Realistic rendering of S186F SARS-COV-2 Nucleocapsid protein (NCp (yellow) and protein 14-3-3 (red and blue) complex.

Figures 3 and 4 are summaries of the docking of phospho-serine 197-NCp and leucine 197-NCp mutant with Protein 14-3-3. Complexes between both phospho-serine 197-NCp and leucine 197-NCp mutant and Protein 14-3-3 are formed, and there was significant change in the conformation of the NCp mutant molecule which was reflected by a large difference in Stability Energy(ΔG_{stability energy}) and Binding Energy (ΔΔG) of the leucine 197-NCp mutant-Protein 14-3-3 complex. The calculated Stability Energy (ΔG_{stability energy}) and Binding Energy (ΔΔG) of phospho-serine 197-NCp-Protein 14-3-3 were calculated to be ~639 Kcal/mol and ~244 Kcal/mol respectively while the calculated Stability Energy (ΔG_{stability energy}) and Binding Energy (ΔΔG_{binding energy}) of leucine 197-NCp mutant-Protein 14-3-3 complex was ~559 Kcal/mol and ~170 Kcal/mol respectively. The Binding Energy Difference (ΔΔΔG_{binding energy difference}) between phospho-serines197 NCp-Protein 14-3-3 complex and leucine 197-NCp mutant-Protein 14-3-3 complex was calculated to be ~ -74 Kcal/mol. These results are consistent with the conclusion that mutation of serine 197 of NCp to leucine was accompanied by destabilization of the NCp-Protein 14-3-3 complex and reduction of the binding affinity between NCp and protein 14-3-3.

Figure 3: A: Ribbon structure (red) of phospho-serine 197-SARS-COV-2 Nucleocapsid protein (NCp) and protein 14-3-3 complex, rendered as described in method section. Phosphorylation rich domain is shown as blue spheres. B: Realistic rendering of phospho-serine 186-SARS-COV-2 Nucleocapsid protein (NCp) (yellow) and protein 14-3-3 pre-complex (Red and Blue).

Figure 4: A: Ribbon structure (red) of S186F-SARS-COV-2 Nucleocapsid protein (NCp) and protein 14-3-3 complex, rendered as described in method section. Blue spheres show the phosphorylation rich domain, B: Realistic rendering of S186F SARS-COV-2 Nucleocapsid protein (NCp (yellow) and protein 14-3-3 (red and blue) complex.

Docking experiments between phospho-serine 202-NCp and asparagine 202-NCp mutant and Protein 14-3-3 are depicted in figures 5 and 6. There was significant change in the conformation of the NCp mutant molecule. The calculated Stability Energy (ΔG_{stability energy}) of phospho-serine 197-NCp-Protein 14-3-3 and asparagine-NCp mutant-Protein 14-3-3 complex were ~574 Kcal/mol and ~499 Kcal/mol respectively while the calculated Binding Energy (ΔΔG_{binding energy}) of phospho-serine 197-NCp-Protein 14-3-3 and asparagine-NCp mutant-Protein 14-3-3 complex were ~184 Kcal/mol and ~112 Kcal/mol respectively. The Binding Energy Difference (ΔΔΔG_{binding energy difference}) between phospho-serine 202 NCp-Protein 14-3-3 complex and leucine asparagine 202-NCp mutant-Protein 14-3-3 complex was calculated to be ~ -72 Kcal/mol. These results are consistent with the conclusion that mutation of serine 202 of NCp to asparagine was accompanied by destabilization of the NCp-Protein 14-3-3 complex and reduction of the binding affinity between NCp and protein 14-3-3.

Figure 5: A: Ribbon structure (red) of phospho-serine 197-SARS-COV-2 Nucleocapsid protein (NCp) and protein 14-3-3 complex, rendered as described in method section. Phosphorylation rich domain is shown as blue spheres. B: Realistic rendering of phospho-serine 186-SARS-COV-2 Nucleocapsid protein (NCp) (yellow) and protein 14-3-3 pre-complex (Red and Blue).

Figure 5: A: Ribbon structure (red) of S186F-SARS-COV-2 Nucleocapsid protein (NCp) and protein 14-3-3 complex, rendered as described in Method Section. Blue spheres show the phosphorylation rich domain, B: Realistic rendering of S186F SARS-COV-2 Nucleocapsid protein (NCp (yellow) and protein 14-3-3 (red and blue) complex.

Table 1 summarizes the thermodynamic calculations of various NCp mutant-Protein 14-3-3 complexes. The results show that mutations of serines 186, 197 and 202, and arginine 203 and glycine 204 were accompanied by decreases in Stability Energy (ΔG_{stability energy}) and Binding Energy (ΔΔG_{binding energy}) indicating that there were enhanced instability of the NCp-14-3-3 complex and lowering of binding affinity between NCp and Protein 14-3-3. Calculation of the Binding Energy Difference (ΔΔΔG_{binding energy difference}) between the wild type and the mutated complexes confirmed these conclusions.

Discussion

Several reports have documented mutations in SARS-COV-2 Nucleocapsid protein (NCp). However, their significance remains to be fully characterized [30-36]. Because of mutations that occur in NCp, Wang et al. [34] have described it as “the worst target of any drug, vaccine, and diagnostic development”. Rahmani et al. [31] appear to agree with Wang et al. [34] as they showed that the double mutation, RG203/204RK destabilized and decreased structural flexibility of NCp. However, the effects of mutations in NCp on its function were only speculated upon. On the other hand, Dutta et al. [37] have proposed that NCp could be an ideal target for vaccine development because they suggested that NCp was mutating at a relatively low rate. The roles of mutations within NCp must be explored and taken into account in any drug or vaccine development program that targets NCp. Furthermore, in view of the fact that multi-sites phosphorylation of NCp affects its function significantly [13-18], any drug or vaccine development program that targets NCp cannot ignore the fact that phosphorylations of NCp can prevent its binding to chemical ligands or antibodies.

It was previously shown that SARS-COV-2 Nucleocapsid protein (NCp) contains a phosphorylation rich domain which forms phosphorylation dependent binding and sequestration sites for Protein 14-3-3 [13,18]. The main phosphorylation dependent and sequestration sites for Protein 14-3-3 consist of the motifs, RNpSTP and RGTpSP (phospho-serines 197 and 206) which are recognized and phosphorylated by C-TAK1, a protein kinase which plays a key role in the control of the cell cycle, [13,18,38-41]. Apart from the sites that are recognized and phosphorylated by C-TAK1, there are other phosphorylation sites, including serine 186, threonine 198, serine 202 and serine 205 [13-18]. Mutations in the phosphorylation sites, serine 186, 197 and 202, and arginine 203 and glycine 204 were identified in strains/substrains isolated from individuals located in various parts of the world. It was proposed that mutations in the phosphorylation sites and phosphorylation recognition motifs allow NCp to evade sequestration by Protein 14-3-3 which would result in enhanced dimerization of NCp, an important step for NCp to act as an essential co-factor for the replication, transcription and packaging of the SARS-COV-2 genome [13,18].

In the present work, through structure model analysis and thermodynamic calculation, we have determined the effects of the above described mutations on the binding and sequestration of NCp by Protein 14-3-3. Our results show that mutations within the phosphorylation domain of NCp, including S186F, S197L, S202N, RG203/204KR and RG203/204KT were accompanied by destabilization and decreased binding efficiency of the components of the NCp mutants-Protein 14-3-3 complexes. The present work provides direct evidence that mutations in NCp affects its dimerization which determines its function as an essential co-factor of replication, transcription and packaging of the SARS-COV-2 genome [1-17].

It is submitted that mutations of the phosphorylation sites, S186F, S197L and S202N and phosphorylation recognition sites, S197L within the phosphorylation recognition motif RNpSTP and, RG203/204KR and RG203/204KT within the phosphorylation motifs, RGTpSP is a mechanism that allows NCp to escape sequestration by 14-3-3 and inhibition of its dimerization. Molecules that act to enhance the sequestration of NCp by Protein 14-3-3 and prevent the dimerization of NCp are potential therapeutics for the control of SARS-COV-2 infection and virulence. Because dimerization of NCp is an essential step for it to act as a co-factor for the replication, transcription and packaging of the SARS-COV-2 genome [1-17], molecules that act to enhance the sequestration of NCp by Protein 14-3-3 and prevent the dimerization of NCp would in principle shut off the replication, transcription and packaging of the SARS-COV-2 genome. One chemical molecule that act to prevent the dimerization of NCp at picomolar concentration has been identified and is being characterized (Limtung, P. and Tung, H.Y.L., manuscript in preparation).

Acknowledgements

This work was supported by the Nacbraht Biomedical Research Institute Fund.

Author Contributions

H.Y. Lim Tung came up with the concept and the questions, performed the experiments with Pierre Limtung, analyzed the results with Pierre Limtung and wrote the paper with Pierre Limtung.

Pierre Limtung performed the experiments with H.Y. Lim Tung, analyzed the results with H.Y. Lim Tung and wrote the paper with Pierre Limtung.

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