Mutation frequency is very high in RNA viruses, which enhances virulence and responsible for the formation of new varieties [19]. mutational analysis of SARS-CoV-2 genomes from different geographical locations, which enabled us to identify numerous unique features of this viral genome. This includes several important country-specific unique mutations in the major proteins of SARS-CoV-2 namely, replicase polyprotein, spike Rabbit Polyclonal to PSMD6 glycoprotein, envelope protein and nucleocapsid protein. Indian strain showed mutation in spike glycoprotein BAY1217389 at R408I and in replicase polyprotein at BAY1217389 I671T, P2144S and A2798V,. While the spike protein of Spain & South Korea carried F797C and S221W mutation, respectively. Likewise, several important country specific mutations were analyzed. The effect of mutations of these major proteins were also investigated using numerous methods. Main protease (Mpro), the restorative target protein of SARS with maximum reported inhibitors, was thoroughly investigated and the effect of mutation around the binding affinity and structural dynamics of Mpro was studied. It was found that the R60C mutation in Mpro affects the protein dynamics, thereby, affecting the binding of inhibitor within its active site. The implications of mutation on structural characteristics were determined. The information provided in this manuscript holds great potential in further scientific research towards the design of potential vaccine candidates/small molecular inhibitor against COVID19. Introduction In the last two decades, three coronaviruses of genus studies were also performed to investigate the effect of mutations around the dynamics of Mpro. The findings of this study provide a clue for the futuristic development of potential vaccine candidate or therapeutic design against COVID19. Material and methods Sequence analysis and stability prediction The genome sequence of ORF1ab for SARS with reference sequence ID: “type”:”entrez-nucleotide”,”attrs”:”text”:”NC_004718.3″,”term_id”:”30271926″,”term_text”:”NC_004718.3″NC_004718.3 and protein sequence with GenBank ID: “type”:”entrez-protein”,”attrs”:”text”:”AAP41036.1″,”term_id”:”30795144″,”term_text”:”AAP41036.1″AAP41036.1, was retrieved from NCBI database. Similarly, the genome sequence for SARS-CoV-2 with Reference Sequence ID: “type”:”entrez-nucleotide”,”attrs”:”text”:”MT012098.1″,”term_id”:”1804119759″,”term_text”:”MT012098.1″MT012098.1, “type”:”entrez-nucleotide”,”attrs”:”text”:”MT019529.1″,”term_id”:”1805293611″,”term_text”:”MT019529.1″MT019529.1, “type”:”entrez-nucleotide”,”attrs”:”text”:”MT039890.1″,”term_id”:”1807860439″,”term_text”:”MT039890.1″MT039890.1, “type”:”entrez-nucleotide”,”attrs”:”text”:”MT093571.1″,”term_id”:”1811294619″,”term_text”:”MT093571.1″MT093571.1, “type”:”entrez-nucleotide”,”attrs”:”text”:”MT192772.1″,”term_id”:”1821109024″,”term_text”:”MT192772.1″MT192772.1, “type”:”entrez-nucleotide”,”attrs”:”text”:”MT126808.1″,”term_id”:”1817836233″,”term_text”:”MT126808.1″MT126808.1, “type”:”entrez-nucleotide”,”attrs”:”text”:”MT192759.1″,”term_id”:”1821108987″,”term_text”:”MT192759.1″MT192759.1, “type”:”entrez-nucleotide”,”attrs”:”text”:”MN985325.1″,”term_id”:”1800408777″,”term_text”:”MN985325.1″MN985325.1, “type”:”entrez-nucleotide”,”attrs”:”text”:”MT007544.1″,”term_id”:”1803016604″,”term_text”:”MT007544.1″MT007544.1, “type”:”entrez-nucleotide”,”attrs”:”text”:”LC529905.1″,”term_id”:”1820247323″,”term_text”:”LC529905.1″LC529905.1, “type”:”entrez-nucleotide”,”attrs”:”text”:”MT020781.2″,”term_id”:”1821514660″,”term_text”:”MT020781.2″MT020781.2, “type”:”entrez-nucleotide”,”attrs”:”text”:”MT072688.1″,”term_id”:”1810678290″,”term_text”:”MT072688.1″MT072688.1 and “type”:”entrez-nucleotide”,”attrs”:”text”:”MT066156.1″,”term_id”:”1809484476″,”term_text”:”MT066156.1″MT066156.1 and protein sequence with GenBank ID: “type”:”entrez-protein”,”attrs”:”text”:”QHS34545.1″,”term_id”:”1804119760″,”term_text”:”QHS34545.1″QHS34545.1, “type”:”entrez-protein”,”attrs”:”text”:”QHU36823.1″,”term_id”:”1805293612″,”term_text”:”QHU36823.1″QHU36823.1, “type”:”entrez-protein”,”attrs”:”text”:”QHZ00378.1″,”term_id”:”1807860440″,”term_text”:”QHZ00378.1″QHZ00378.1, “type”:”entrez-protein”,”attrs”:”text”:”QIC53203.1″,”term_id”:”1811294620″,”term_text”:”QIC53203.1″QIC53203.1, “type”:”entrez-protein”,”attrs”:”text”:”QIK50437.1″,”term_id”:”1821109025″,”term_text”:”QIK50437.1″QIK50437.1, “type”:”entrez-protein”,”attrs”:”text”:”QIG55993.1″,”term_id”:”1817836234″,”term_text”:”QIG55993.1″QIG55993.1, “type”:”entrez-protein”,”attrs”:”text”:”QIK50416.1″,”term_id”:”1821108988″,”term_text”:”QIK50416.1″QIK50416.1, “type”:”entrez-protein”,”attrs”:”text”:”QHO60603.1″,”term_id”:”1800408787″,”term_text”:”QHO60603.1″QHO60603.1, “type”:”entrez-protein”,”attrs”:”text”:”QHR84448.1″,”term_id”:”1803016605″,”term_text”:”QHR84448.1″QHR84448.1, “type”:”entrez-protein”,”attrs”:”text”:”BCB15089.1″,”term_id”:”1820247324″,”term_text”:”BCB15089.1″BCB15089.1, “type”:”entrez-protein”,”attrs”:”text”:”QHU79171.2″,”term_id”:”1821514661″,”term_text”:”QHU79171.2″QHU79171.2, “type”:”entrez-protein”,”attrs”:”text”:”QIB84672.1″,”term_id”:”1810678291″,”term_text”:”QIB84672.1″QIB84672.1 “type”:”entrez-protein”,”attrs”:”text”:”QIA98553.1″,”term_id”:”1809484477″,”term_text”:”QIA98553.1″QIA98553.1 for India, China, South-Korea, Sweden, Vietnam, Brazil, Taiwan, USA, Australia, Japan, Finland, Nepal and Italy, respectively, were downloaded BAY1217389 from NCBI. Multiple sequence alignment (MSA) and visualization of SARS and SARS-CoV-2 sequences from 13 different countries was performed using Molecular Evolutionary Genetics Analysis (MEGA) version 10.1.8. To delineate and analyze the mutation across different countries, an in-house script written in Perl and Python was used. MUPRO server was used to determine the effect of mutation on various SARS-CoV-2 proteins [21]. Model building The crystal structure of Mpro protein from SARS-CoV-2 in complex with Boceprevir (pdb id: 7BRP) was taken as a wildtype (WT). The structure of R60C was generated by inserting the Point mutation and modelled using modeler 9v13 [22]. Molecular docking Boceprevir was retrieved and redocked within the structure of Mpro using CCDC Gold. The RMSD for the crystal and redocked conformation of Boceprevir were compared. Further, Boceprevir was subjected to dock within the active site of R60C mutant. The poses were visualized on PMV viewer [23]. Molecular dynamics simulations of the protein and their complexes The structure of Boceprevir in complex with Mpro (WT) and R60C mutant was subjected to energy minimization using Gromacs-5 with the CHARMM27 all atom pressure field [24C26]. The models were solvated with a SPC/E water model in a cubic periodic box with 1 nm distance from the edge of the complex atoms. The solvated system was neutralized by seven chloride ions. The system was, thereafter, minimized using steepest descent algorithm with convergence criteria of tolerance value 1000 kJ mol?1 nm?2. The complete simulation of minimized solvated proteins was performed under periodic boundary condition with time step of 2 fs. Particle mesh Ewald was used for long range electrostatic interactions with an interpolation order of 4 and a Fourier spacing of 0.16. The first phase simulation was conducted under an NVT ensemble for 500 ps by keeping all bonds constrained using the LINCS algorithm for heat equilibration. The system was heated to 300 K using leap-frog integrator while pressure coupling was set off. A V-rescale thermostat was used to maintain constant heat for each system, followed by pressure equilibration at 300 K using Parrinello-Rahman pressure coupling algorithm under an isothermal-isobaric ensemble for another 500 ps at 1.0 bar. Isothermal compressibility of the solvent was set to 4.5e-5 bar?1. Further, simulation of 50000 ps production run was carried out at 300 K and 1 atm BAY1217389 pressure for trajectory analysis. The final models obtained at the end of MD were validated.