Mapping mutations in proteins of SARS CoV-2 Indian isolates on to the threedimensional structures
The amino acid residue mutations observed in SARS CoV-2 RNA dependent RNA polymerase, helicase, endoRNAse and spike proteins from Indian isolates, relative to the reference SARS CoV-2 proteins from the Wuhan Hu-1 isolate from China, were mapped onto the protein three-dimensional structure templates available in the Protein Data Bank. The secondary structure conformations corresponding to the mutations, their locations and proximity to functionally important residues in the proteins and to the drug binding sites in RNA dependent RNA polymerase and endoRNAse targets were analysed. Our analyses provide structural insights into the mutations in these SARS CoV-2 proteins.
Introduction:<!>Materials and Methods:<!>Results and Discussion:<!>Mapping the mutation sites in RNA-dependent RNA polymerase (Nsp12)<!>Mutation sites in helicase (Nsp13)<!>Mutation sites in the spike (S) glycoprotein<!>Conclusions:
<p>The current COVID-19 pandemic disease that has resulted in over 11 million coronavirus cases and above half a million deaths worldwide (https://www.worldometers.info/coronavirus/), seriously awaits intervention in the form of vaccine and drugs for the prevention and treatment of this deadly disease. Several organizations and research groups in the world are currently engaged in this endeavour to come up with a successful therapeutic treatment. A successful vaccine depends upon its utility over populations worldwide. Mutations in SARS CoV-2 spike glycoproteins from several infected individuals representing different countries have been reported and the mutations in the spike protein have been mapped onto the protein structure (Korber et al., 2020). Also, two mutations in the spike protein of the first two SARS-CoV-2 sequences from India are mapped on to protein structure (Yadav et al., 2020). Based on the analysis of protein sequences from SARS CoV-2 representing 22 Indian isolates available in the publicly accessible NCBI databank (http://www.ncbi.nlm.nih.gov), we identified not only certain new mutations in the spike protein of SARS CoV-2 among the Indian isolates but also mutations in ten additional SARS CoV-2 proteins (Guruprasad, 2020).</p><p>In this work, we analyse the secondary structure conformations and map the mutation site residues for SARS CoV-2 proteins observed in the Indian isolates on to the protein threedimensional structures of RdRp, helicase, endoRNAse and spike proteins for which suitable templates are available in the Protein Data Bank (PDB). We further analyse the proximity of the mutation sites to functionally important residues in the protein three-dimensional structure and to the Remdesivir and Tipiracil drug binding sites in RdRp and endoRNAse proteins, respectively.</p><!><p>The structural templates for mapping the mutation sites of the SARS CoV2 proteins in the Indian isolates were obtained by searching the Protein Data Bank (PDB) (Berman et al., 2000) (http://www.rcsb.org) with the PSI-BLASTP program (Schäffer et al., 2001) available at the NCBI website (http://www.ncbi.nlm.nih.gov). The reference protein sequences of SARS CoV-2 Wuhan-Hu-1 isolate were used as the query. The secondary structure conformations were inferred from PDBsum (Laskowski et al., 2001) (https://www.ebi.ac.uk/pdbsum). The PyMol software (DeLano, 2002) was used for molecular visualization and structure superposition.</p><!><p>Among the eleven proteins; Nsp2, Nsp3, Nsp4, Nsp6, RNA-dependent RNA polymerase (RdRp), helicase, endoRNAse, spike glycoprotein, nucleocapsid phosphoprotein 'N', orf3a and orf8 proteins associated with mutations in SARS CoV-2 Indian isolates (Guruprasad, 2020), suitable structural templates to map the mutations were available for the SARS CoV-2 RdRp, endoRNAse, spike and helicase proteins. The secondary structure conformations associated with the mutation site residues in the protein structures are shown in Table 1.</p><!><p>The RdRp (or Nsp12) protein plays a central role in replication and transcription cycle of the virus by catalyzing the synthesis of viral RNA with assistance of cofactors (Subissi et al., 2014). The protein with PDB code: 6M71 was selected as template for RdRp that corresponds to the electron microscopy structure of human SARS CoV-2 RdRp in complex with cofactors;</p><p>Nsp7, Nsp8 determined at 2.9Å resolution (Gao et al., 2020). The overall fold of the protein complex is shown in Figure 1  and Phe48 in the β-hairpin (magenta surface) make contacts with residues; Lys121, Tyr122, Asp126, Tyr129, Thr206, Asp208 and Ser236 in NiRAN domain (cyan surface) and with residues; Asp711, Asn713, Tyr728, Arg733, His725 and Glu729 in palm sub-domain (orange surface) and these interactions are important for stability of the overall protein structure (Gao et al., 2020). It can be seen from Figure 3, that none of the residues at the mutation sites (red spheres) are close to the residues at interaction interface of β-hairpin with the NiRAN domain or β-hairpin with palm sub-domain that could affect overall stability. We infer from Figure 4, that mutation of leucine to isoleucine at position 329 would not affect the hydrogen bond involving the main-chain atoms of Leu329 and Thr344. Remdesivir drug inhibits virus proliferation and is suggested as a potential treatment for COVID-19 viral infections with clinical potential (Wang et al., 2020, Holshue et al., 2020). Based on the superposition of the electron microscopy structure of human SARS CoV-2 RdRp in complex with cofactors (PDB code: 6M71) (magenta) and the electron microscopy structure of the Nsp12-Nsp7-Nsp8 complex bound to the template primer RNA and triphosphate form of Remdesivir (PDB code: 7BV2) (green) (Yin et al., 2020), we note that the SARS CoV-2 Indian isolate mutation site residues are distant from the Remdesivir drug binding site in the RdRp protein as shown in Figure 5. The protein is excluded for the sake of clarity and the mutation site residues (red spheres) are shown in Figure 6, along with the zinc ions (green spheres) and magnesium ions (purple spheres) as in the crystal structure. The side-chains of residues in the polymerase domain important for the functional activity and residues that interact with the β-hairpin required for maintaining overall stability of the structure are also shown.</p><p>Ala97 which is in a loop conformation is present immediately after a type I beta-turn and is located near the protein surface. Ala185 is in the middle of helix. Ile201 is at the beginning of a beta-strand near the protein surface and its side-chain makes close contacts with side-chains of Ile86 and Leu90 on a neighbouring helix. Pro323 is at the start of a G-type helix near the protein surface and at the junction between helices; H13 and H15. Leu329 is associated with a twisted anti-parallel beta-strand and is near the protein surface with side-chain close to Cys114, Pro116, Gly345, His347 on neighbouring beta-strands. Ala406 is near the protein surface and present on a long loop between Ala400-Asn414. Thr644 is associated with i+2 nd position or equivalent i+1 th position of overlapping type IV beta-turns and its side-chain is exposed to the protein surface. Val880 is associated with helix (H39) located near the protein surface and its side-chain makes contacts with Pro834 and Leu838 on a neighbouring helix located within the interior of a core formed by four helices (H36, H37, H39 and H18).</p><p>In summary, the SARS CoV-2 mutation sites in the RdRp protein are associated with loops, helices, beta-strand, beta-turns and located near the protein surface. The mutation site residues are distant from the functionally important residues in the protein, and from the interface residues of β-hairpin interactions with the NiRAN and palm domains that is required for maintaining stability of the overall structure and also from the Remdesivir drug binding site.</p><p>The main-chain hydrogen bond involving the residue at position 329 in the RdRp protein of SARS CoV-2 Indian isolate will not be affected due to the L329I mutation.</p><!><p>The helicase SARS CoV (Nsp13) protein catalyzes the unwinding of duplex oligonucleotides into single strands in an NTP-dependent manner and its activity is enhanced by the SARS-Nsp12 protein (Jia et al., 2019). The protein with PDB code: 6JYT was used as template for mapping the mutations and represents the crystal structure of the full-length SARS-Nsp13 protein upon ATP hydrolysis determined at 2.8Å resolution with the overall fold in the shape of a pyramid (Jia et al., 2019). The SARS-Nsp13 protein fold is shown in Figure 7 and it comprises five domains; the 1A (magenta), 2A (cyan) domains, 1B domain (yellow) at the base, ZBD (green) at the apex and S domain (blue) connecting the ZBD domain to the base of the pyramid. The S166A and T214I mutation sites are associated with the 1B domain and P504L mutation is associated with the 2A domain and the side-chains of all three residues are exposed near the protein surface. Ser166 is located close to end of a beta-strand and T214 is at the i+1 th position of a type IV beta-turn. P504 lies within a 14-residue loop (VREFLTRNPAWRKA) essentially comprising beta and gamma turns and is associated with the i+1 th or equivalent i th position of overlapping type IV beta-turns. None of the mutations are close to any of the functionally important residues; R337, R339, K345 and K347 in the β19-β20 loop (tint) in 1A domain as shown in Figure 8, or to the six residues; Lys288, Ser289, Asp374, Glu375, Gln404, Arg567 in the 1A and 2A domains (shown in purple and cyan, respectively), in Figure 9, or to the ZBD domain in SARS-Nsp13 which interacts with SARS-Nsp12. The side-chain atom (NH1) of Arg337 (3.2Å) is involved in hydrogen bond with mainchain carbonyl oxygen atom (C=O) of T214 in the 1B domain and the T214I mutation will not affect this hydrogen bond interaction.</p><p>In summary, mutations in the SARS CoV-2 Indian isolate helicase protein are associated with beta-strand and beta-turns near the protein surface and distant from functionally important residues in the protein.</p><!><p>The trans-membrane spike (S) glycoprotein forms homotrimers protruding from the viral surface and mediates coronavirus entry into host cells (Tortorici and Veesler, 2019). Three 6ACG) as the region corresponding to the mutation site is not available in the SARS CoV2 structure. A930 is in the S2 subunit (Yadav et al., 2020) and is close to the mutational cluster that possibly impacts the fusion core of the first heptad repeat region HR1 (Korber et al., 2020).</p><p>A930 is present in the middle of helix (H15) comprising 21 amino acid residues (Q920KLIANQFNSAIGKIQDSLSS940) (PDB code: 6VXX_A-chain, green) with side-chain pointing towards Val722 on a neighbouring twisted beta-strand S711IAIPTNFTISVTTEILP728</p><p>(blue) as shown in Figure 10C. The H15 helix immediately follows a short helix (H14); N914VLYE918 (orange) and Asn919 is at the 'kink' region between the two helices close to the glycosylation site NAG-1312. Ala930Val mutation in human SARS CoV-2 is in a position to make favourable hydrophobic contacts with the side-chain of Val722. The D614G mutation is associated with the S1 subunit of spike glycoprotein (Korber et al., 2020). D614 is the first residue in a loop immediately at end of a beta-strand associated with a motif comprising 5 anti-parallel beta-strands in the beta-sheet 'E' (PDB code: 6VXX) and is close to the glycosylation binding motif N616CT near NAG-1309 (Figure 10D). The Cys617 residue in the loop makes disulfide bridge with Cys649 on a beta-strand in the beta-sheet ('E') and the sidechain of Asp614 is oriented towards the protomer 'B' (shown in cyan) in the spike glycoprotein. The D614G mutation site is distant from the RBD. Cys1250 is associated with the cytoplasmic region of the spike glycoprotein and a suitable template was not available to map this region onto the protein structure. Q271 is associated with the S1 subunit and is located at the start of the 2 nd beta-strand in a motif comprising 3 anti-parallel beta-strands in the betasheet 'C' (shown in orange) with its side-chain (shown as spheres) exposed on the protein surface (Figure 10E). R408 is associated with the RBD in S1 subunit (Yadav et al, 2020). In the crystal structure of the RBD of spike protein (cyan) bound to the ACE-2 receptor (green) (PDB code: 6LZG), a long curved helix comprising 32 amino acid residues; Ile21-Thr52 the SARS CoV-2 NendoU hexamer made up of dimer of trimers and that is essential for the enzymatic activity (Kim et al., 2020a) was generated using the PISA server available at https://www.ebi.ac.uk/msd-srv/prot_int/cgi-bin/piserver and the mutations were also mapped onto the hexamer. The active sites in the protein within the hexamer are located at the top and bottom of the assembly (Kim et al., 2020a) as shown in Figure 12 and the sites of the mutations are distant from the active sites also within the hexamer. The NendoU monomers interact extensively with all five subunits of the hexamer rendering it susceptible to mutations that affect the oligomeric assembly (Kim et al., 2020). In Figure 13, the model of the NendoU hexamer is shown with monomer (orange) making contacts with other five monomers (green, magenta, yellow, blue and cyan) and the three mutation sites; Val149, Asp268, Asp273 are also shown (red spheres) for the monomer (orange). The mutation sites are distant from the interface sites of inter-monomer interactions in the hexamer. Further, it was observed that the mutation site residues are distant from the site where Tipiracil drug (5-chloro-6-[(2iminopyrrolidin-1-yl)methyl]-1H-pyrimidine-2,4-dione) binds to the protein as observed in the crystal structure complex of the Nsp15 SARS CoV-2 complex (PDB code: 6WXC_Achain) (Kim et al., 2020b) and shown in Figure 14.</p><p>In summary, the residues at the three mutation sites in Nsp15 protein of human SARS CoV-2</p><p>Indian isolates are associated with beta-turns or loops in the protein structure and the sidechains are exposed to the protein surface. The mutation sites are distant from the functionally important residues, inter-monomer interaction interfaces in the hexamer and from the Tipiracil drug-binding site in the SARS CoV-2 Nsp15 protein.</p><p>We could not find suitable templates to map the mutations for the Nsp2, Nsp3, Nsp4, Nsp6, orf3a, orf8 proteins or the nucleocapsid phosphoprotein 'N' (Guruprasad, 2020).</p><!><p>The In summary, mutations analysed for the RdRp, helicase, spike and endoRNAse proteins among the twenty-two SARS CoV-2 Indian isolates are situated at locations that are generally distant or that do not affect the functionally important residues in the protein threedimensional structure.</p>