key: cord-0959582-v05h35jn authors: Shafiq, Athar; Zubair, Farrukh; Ambreen, Amna; Suleman, Muhammad; Yousafi, Qudsia; Rasul Niazi, Zahid; Anwar, Zeeshan; Khan, Abbas; Mohammad, Anwar; Wei, Dong-Qing title: Investigation of the binding and dynamic features of A.30 variant revealed higher binding of RBD for hACE2 and escapes the neutralizing antibody: A molecular simulation approach date: 2022-04-30 journal: Comput Biol Med DOI: 10.1016/j.compbiomed.2022.105574 sha: 0d1e9b090cb24d903ffb816ecba5181bdc0dd741 doc_id: 959582 cord_uid: v05h35jn With the emergence of Delta and Omicron variants, many other important variants of SARS-CoV-2, which cause Coronavirus disease-2019, including A.30, are reported to increase the concern created by the global pandemic. The A.30 variant, reported in Tanzania and other countries, harbors spike gene mutations that help this strain to bind more robustly and to escape neutralizing antibodies. The present study uses molecular modelling and simulation-based approaches to investigate the key features of this strain that result in greater infectivity. The protein-protein docking results for the spike protein demonstrated that additional interactions, particularly two salt-bridges formed by the mutated residue Lys484, increase binding affinity, while the loss of key residues at the N terminal domain (NTD) result in a change to binding conformation with monoclonal antibodies, thus escaping their neutralizing effects. Moreover, we deeply studied the atomic features of these binding complexes through molecular simulation, which revealed differential dynamics when compared to wild type. Analysis of the binding free energy using MM/GBSA revealed that the total binding free energy (TBE) for the wild type receptor-binding domain (RBD) complex was −58.25 kcal/mol in contrast to the A.30 RBD complex, which reported −65.59 kcal/mol. The higher TBE for the A.30 RBD complex signifies a more robust interaction between A.30 variant RBD with ACE2 than the wild type, allowing the variant to bind and spread more promptly. The BFE for the wild type NTD complex was calculated to be −65.76 kcal/mol, while the A.30 NTD complex was estimated to be −49.35 kcal/mol. This shows the impact of the reported substitutions and deletions in the NTD of A.30 variant, which consequently reduce the binding of mAb, allowing it to evade the immune response of the host. The reported results will aid the development of cross-protective drugs against SARS-CoV-2 and its variants. RNA-dependent RNA polymerase (RDRP) [1, 2] . 58 The coronavirus infection in cells begins when the spike protein binds to the host 59 angiotensin-converting enzyme 2 (ACE2). Human ACE2 (hACE2) is mostly expressed in the 60 lungs, kidneys, and small intestine, which might result in significant sickness [3] . After binding 61 with ACE2, the host cell proteases split the SARS-COV-2 S-protein into the S1-ectodomain 62 and S2 membrane-anchored domain, which are located at the N-terminal and C-terminal 63 respectively. The S1 subunit aids in the recognition of cell surface receptors as well as The SARS-CoV-2 are evolutionary gamblers that lacks a proofreading capabilities thus 71 prone to enormous mutation frequency (million times > DNA-containing cells). Many variants 72 of the SARS-CoV-2 virus have now been detected, as part of the ongoing worldwide pandemic, 73 and have been associated with rapid transmissibility, evasion of immune responses, high 74 occurrence of indisposition, and recurrence [11] . There are several variants of concern (VOC) concern for public health response. The A.30 variant has 5 deletion and 10 substitution 94 mutations in the N terminal domain of the S1 protein, which acts as an antigenic epitope and 95 can be bound by neutralizing antibodies [17] . Furthermore, three mutations were discovered in 96 the RBD, which interacts with the human ACE2 receptor and is a primary target of antibodies 97 for neutralization. T478R and E484K are two of these mutations, occurring at the ACE2 98 binding site; however, the letter is responsible to develop resistance to antibody-mediated The spike protein, which is required for viral interaction with the host cell, was reported to 115 have mutations in emerging strains. The recently submitted SARS-CoV-2 amino acid sequence 116 was retrieved from the UniProt database (accession number P0DTC2) for identification of 117 mutation loci in newly evolved variants [19] . The wild type protein structure of SARS-CoV-2 118 spike protein (6M0J) was retrieved from the PDB database and AlphFold2 was used to model 119 the predicted mutations in the wild type protein structure [20, 21] . The NTD and mAb 120 structures were also collected from RCSB using 7C2L accession number [20] . Binding network and dissociation constant (KD) determination 123 The HADDOCK (high ambiguity-driven protein-protein docking) online server was used to 124 investigate the binding differences between the wild type and novel strain A.30 RBD and NTD The molecular dynamics behavior of both wild type and mutant complexes was analyzed with 141 the AMBER20 package, which uses the FF19SB force field [26-28]. The system was 142 neutralized by 23 sodium ions added to each system and the system solvation was performed 143 by using the OPC (optimal point charge) water box model (9180 water molecules). Afterward, The CPPTRAJ package of AMBER20 was used to analyze trajectories and CUDA was used For the wild type RBD-ACE2 complex, the docking score was predicted to be -111. additional interactions were also seen in the A.30 RBD-ACE2 complex only. As given in Table 1 . Figure 3A and 3B. The docking scores for each complex are given in Table 282 1. The binding strength of the two A.30 complexes, RBD-ACE2 and NTD-mAb, was estimated 289 through KD prediction which has been previously used for other variants such as B. and NTD, and the A.30 RBD and NTD complexes are given in Table 1 . J o u r n a l P r e -p r o o f To validate the RMSD findings, radius of gyration (Rg) analysis was performed to examine 340 the system's compactness over time ( Figure 4B) . As with RMSD, Rg illustrated that the wild NTD-mAb complex was reported to be 0.9 Å. The RMSD for the A.30 NTD-mAb complex 395 continued to increase gradually but reported more structural perturbation than the wild type. The average RMSD increased during the last 150 ns and was calculated to be 1.10 Å. Previously, similar findings were reported for other variants, such as B. The radius of gyration for both the complexes reported a strong agreement with the RMSD 403 results. As can be seen in Figure 6B , the simulation trajectory, hydrogen bonding analysis was performed. As given in Figure 6C , 416 the wild type reported more hydrogen bonds than the A.30 NTD-mAb complex. In the wild for each complex is given in Table 2 . Residue Flexibility Analysis of NTD 430 We also predicted the local level residue flexibility for the NTD of wild type and A.30 variants 431 in complex with mAb. As shown in Figure 7 , the wild type NTD is comparatively more stable The MM/GBSA technique for computing the BFE of biological partners is a widely used 444 method for examining the putative docking configuration. This method, which is less costly 445 than the alchemical free energy methods, displays the binding stability of interacting key 446 regions and the BFE. It is also regarded to be more precise than any rational scoring function. 447 We employed the MM/GBSA technique because it allows us to see how the mutations in the 448 spike RBD influence the binding with hACE2 and NTD with mAb. The BFE results are given 449 in Table 3 . 450 Binding Free energy for RBD-ACE2 complexes 451 As given in In conclusion, the current study employed protein-protein coupling and molecular simulation 473 approaches to decipher the key features required for stronger interaction with the ACE2 and The computations were partially performed at the PengCheng Lab. and the Center for High-490 Performance Computing, Shanghai Jiao Tong University. Availability of data and material 493 All the data is available on RCSB, UniProt and any simulation data would be provided on reasonable 494 demand. The accession numbers to access this data are given in the manuscript. Ethics approval and consent to participate 497 Coronavirus biology and replication: 507 implications for SARS-CoV SARS-CoV-2 variants and 509 ending the COVID-19 pandemic 511 Temporal signal and the phylodynamic threshold of SARS-CoV-2, Virus evolution Mechanisms of Coronavirus Cell Entry 513 Mediated by the Viral Spike Protein, Viruses Structural basis for the recognition of SARS-CoV-2 515 by full-length human ACE2 Structural insights into coronavirus entry 519 Evolutionary and structural analysis of SARS-CoV-2 specific evasion of host immunity ADP-ribose) glycohydrolase (PARG) 524 structures with inhibitors Structural, biophysical, and 526 biochemical elucidation of the SARS-CoV-2 nonstructural protein 3 macro domain Structural genomics of SARS-CoV-2 indicates evolutionary conserved functional regions of viral 530 proteins The biological and clinical significance of emerging SARS-CoV-2 variants What's important to know about SARS-CoV-2 variants of 535 concern? Recurrent emergence of SARS-CoV-2 spike deletion H69/V70 and its role in the 538 Alpha variant B. 1.1. 7 Variants of SARS-CoV-2 SARS-CoV-2 B. 1.617. 2 Delta variant replication and immune evasion The Omicron (B. 1.1. 529) variant of SARS-CoV-2 binds to the hACE2 receptor more 545 strongly and escapes the antibody response: Insights from structural and simulation data N-551 terminal domain antigenic mapping reveals a site of vulnerability for SARS-CoV-2 The spike protein of 555 SARS-CoV-2 variant A.30 is heavily mutated and evades vaccine-induced antibodies with high 556 efficiency UniProt Knowledgebase: a hub of integrated protein data A 560 neutralizing human antibody binds to the N-terminal domain of the Spike protein of SARS-CoV-2, 561 Highly accurate protein structure prediction with AlphaFold HADDOCK: a protein− protein docking approach based 566 on biochemical or biophysical information Shape-restrained modelling of protein-small molecule 569 complexes with HADDOCK, bioRxiv PDBsum: summaries and analyses of PDB structures PRODIGY: a web server for 573 predicting the binding affinity of protein-protein complexes The Amber biomolecular simulation programs AMBER, a package of computer programs for applying molecular mechanics, 579 normal mode analysis, molecular dynamics and free energy calculations to simulate the structural 580 and energetic properties of molecules An overview of the Amber biomolecular simulation 582 package Steepest descent A stable, rapidly converging conjugate 586 gradient method for energy minimization Routine microsecond 588 molecular dynamics simulations with AMBER on GPUs. 2. Explicit solvent particle mesh Ewald CPPTRAJ: software for processing and analysis of 591 molecular dynamics trajectory data Deep-learning-based target screening and 594 similarity search for the predicted inhibitors of the pathways in Parkinson's disease, RSC advances Pyrazinamide 597 resistance of novel mutations in pncA and their dynamic behavior Structural Insights 600 into the mechanism of RNA recognition by the N-terminal RNA-binding domain of the SARS-CoV-2 601 nucleocapsid phosphoprotein Structural probing of HapR to identify potent phytochemicals to control Vibrio cholera 604 through integrated computational approaches The systematic modeling 606 studies and free energy calculations of the phenazine compounds as anti-tuberculosis agents De novo design of novel protease 609 inhibitor candidates in the treatment of SARS-CoV-2 using deep learning, docking, and molecular 610 dynamic simulations VARIDT 2.0: structural variability of drug transporter Exploring the Binding 615 Mechanism of Metabotropic Glutamate Receptor 5 Negative Allosteric Modulators in Clinical Trials 616 by Molecular Dynamics Simulations Tracking the interaction between single-wall carbon 618 nanotube and SARS-Cov-2 spike glycoprotein: A molecular dynamics simulations study Norepinephrine Reuptake Inhibitors' Dual-Targeting Mechanism? The Key Role of Transmembrane 622 Domain 6 in Human Serotonin and Norepinephrine Transporters Revealed by Molecular Dynamics 623 Simulation Molecular Mechanism for the Allosteric Inhibition 625 of the Human Serotonin Transporter by Antidepressant Escitalopram Bioinformatics analysis of the differences in the binding profile of the wild-type and mutants of the 629 SARS-CoV-2 spike protein variants with the ACE2 receptor The SARS-CoV-2 B.1.618 variant 633 slightly alters the spike RBD-ACE2 binding affinity and is an antibody escaping variant: a 634 computational structural perspective Higher 636 infectivity of the SARS-CoV-2 new variants is associated with K417N/T, E484K, and N501Y mutants: 637 An insight from structural data The 639 SARS-CoV-2 B. 1.618 variant slightly alters the spike RBD-ACE2 binding affinity and is an antibody 640 escaping variant: a computational structural perspective Interactions of the Receptor Binding Domain of SARS-CoV-2 Variants with hACE2: Insights 643 from Molecular Docking Analysis and Molecular Dynamic Simulation Higher 645 infectivity of the SARS-CoV-2 new variants is associated with K417N/T, E484K, and N501Y mutants: 646 An insight from structural data Why Does the Novel Coronavirus Spike Protein 648 Interact so Strongly with the Human ACE2? A Thermodynamic Answer Deep mutational scanning of SARS-CoV-2 receptor binding domain 652 reveals constraints on folding and ACE2 binding Somatic 655 Hypermutation-Induced Changes in the Structure and Dynamics of HIV-1 Broadly Neutralizing Role of framework 658 mutations and antibody flexibility in the evolution of broadly neutralizing antibodies The spike protein of SARS-CoV-2 variant A. 30 is heavily mutated and evades 662 vaccine-induced antibodies with high efficiency Inhibition of SARS-CoV-2 665 (previously 2019-nCoV) infection by a highly potent pan-coronavirus fusion inhibitor targeting its 666 spike protein that harbors a high capacity to mediate membrane fusion Regulation of protein-ligand 669 binding affinity by hydrogen bond pairing Entropy-enthalpy compensation: role and ramifications in 671 biomolecular ligand recognition and design, Annual review of biophysics Optimized hydrophobic 673 interactions and hydrogen bonding at the target-ligand interface leads the pathways of drug-674 designing 1607-1618. the emergence of Delta and Omicron variants, many other important variants of SARS-CoV-2 including A Keeping in view the required data the current study uses molecular modelling and simulation-based approaches to investigate the key features required for higher infections • The protein-protein docking results demonstrated that additional interactions particularly the two salt-bridges by the mutated residue Lys484 help in the higher binding while the loss of key residues at NTD helps the virus to bind mAb in a we deeply studied the atomic features through molecular simulation which revealed differential dynamics when compared to the wild type This shows the impact of the reported substitutions and deletions in the NTD of A.30 variant which consequently reduces the binding of mAb towards NTD and evades the immune response instigated by the host • The reported finding will aid the development of cross-protective drugs against the SARS-CoV-2 and its variants J o u r n a l P r e -p r o o f Declaration of Interest: Authors declare there is no declaration of interest.