key: cord-0277980-bzzkk1gn authors: Gupta, Aayatti Mallick; Chakrabarti, Jaydeb title: Effect on the conformations of spike protein of SARS-CoV-2 due to mutation date: 2022-05-12 journal: bioRxiv DOI: 10.1101/2022.05.11.491583 sha: ef0c3018c4c4d56a259f853beb91004907bd3a94 doc_id: 277980 cord_uid: bzzkk1gn The spike protein of SARS CoV-2 mediates receptor binding and cell entry and is the key immunogenic target for virus neutralization and the present attention of many vaccine layouts. It exhibits significant conformational flexibility. We study the structural fluctuations of spike protein among the most common mutations appeared in variant of concerns (VOC). We report the thermodynamics of conformational changes in mutant spike protein with respect to the wildtype from the distributions of the dihedral angles obtained from the equilibrium configurations generated via all-atom molecular dynamics simulations. We find that the mutation causes the increase in distance between N-terminal domain and receptor binding domain leading to an obtuse angle cosine θ distribution in the trimeric structure in spike protein. Thus, increase in open-state is conferred to the more infectious variants of SARS-CoV-2. The thermodynamically destabilized and disordered residues of receptor binding motif among the mutant variants of spike protein are proposed to serve as better binding sites for host factor. We identify a short stretch of region connecting the N-terminal domain and receptor binding domain forming linker loop where many residues undergo stabilization in the open state compared to the closed one. Since the first documented cases of SARS-CoV-2 1 infection in Wuhan, China in late 2019 2 , the COVID-19 pandemic poses an unprecedented threat to the global public health, with more than 286 million infections and over 5.4 million deaths around the world (https://www.who.int/emergencies/diseases/novel-coronavirus-2019/situation-reports/). Despite rapid development and emergency authorization of vaccines, immune escape mutants have emerged, and SARS-CoV-2 infections remain a concern for the global community. Although vaccination has significantly lowered the rates of hospitalization, severity and death [3] [4] [5] [6] , current vaccines do not confer absolute prevention of upper airway transmission of SARS-CoV-2. The numbers of vaccine breakthrough infections and re-infections, consequently, have been continuously reported [7] [8] [9] . It is of vital importance to examine the impact of the mutant variants as soon as they are detected by genomic sequence analysis. In the light of crucial role of spike protein in virus infection and host immune evasion, studies have been prioritized on the emerging mutations of spike protein circulating SARS-CoV-2 strains and investigations on their biological significance 10 . The structural information is essential for structure-based design of vaccine immunogens and entry inhibitors of SARS-CoV-2. Spike protein (180-200 kDa) of SARS-CoV-2 virus consists of a homo-trimeric large clovershaped protrusion that mediates viral entry to the host cell through the human ACE2 receptor 11 distributed mainly in the lung, intestine, heart, and kidney, and alveolar epithelial type II cells 12 . Each spike monomer (1273 aa) consists of a signal peptide located at the N-terminus, the S1 subunit and the S2 subunit. The S1 subunit is responsible for receptor binding comprise of an N-terminal domain (NTD) and a receptor-binding domain (RBD). A short stretch of amino acid residues connects the NTD arm with that of the RBD forms the linker. The S2 subunit comprises of the fusion peptide (FP), heptapeptide repeat sequence 1 (HR1), heptapeptide repeat sequence 2 (HR2), transmembrane (TM) domain and cytoplasm domain. The S2 domain entangles to create the stalk, transmembrane, and small intracellular domains 13 . Spike protein subsists in a metastable, prefusion conformation acting as an inactive precursor. However, when the virus interacts with the host cell, extensive structural rearrangement of the spike protein occurs, by cleaving it into S1 and S2 subunits, allowing the virus to fuse with the host cell membrane. RBD located in the S1 subunit interacts with the cell receptor ACE2 in the region of aminopeptidase N. Remarkable conformational heterogeneity can be found in the RBD region. [16] [17] [18] [19] [20] [21] [22] [23] . It has been observed that alpha, beta and gamma variants contain some common mutations, like E484K and N501Y in the receptor binding motif of RBD region. N501Y and E484K have been reported for increase in ACE2 binding affinity 24 and decrease in efficacy for antibody binding accountable for immune evasion 25, 26 . Thus, particularly the RBD variants in SARS-CoV-2 are vital to recognize mutant viral strains with higher transmissibility and the potentiality to bring about immune invasion. Here we study the effect of mutations in RBD on the conformations of spike protein of SARS-CoV-2. We are particularly interested in the stability of the mutated protein with respect to the wild type. The relative stability of protein conformations has been extracted from mean field description based on conformational thermodynamics data [27] [28] [29] [30] . In this method, the changes in thermodynamics free energy and entropy of a protein in a conformation with respect to a reference conformation are estimated from fluctuations of the dihedral angles in the two states over the simulated trajectories. Earlier studies based on conformational thermodynamics suggest that the destabilized and disordered residues of a protein in a particular conformation are the functional ones in that state, leading to binding specificity 30 . We perform all atom MD simulations of the complete spike protein of SARS-CoV-2 in its wild type and the several mutated VOC strains, like K417N, L452R, E484K and N501Y using the GROMOS96 53a6 force-field in the GROMACS 2018.6 package. we find that the spike protein prefers to be in open state under mutations in the RBD, while a close conformation is preferred in the wild type. We calculate the thermodynamics cost of conformational changes. The RDB residues in mt(K417N)-spike, mt(L452R)-spike, mt(E484K)-spike, mt(N501Y)-spike and mt(mult)-spike show destabilization and disorder with respect to the wild type conformation. On the other hand, the NTD region does not reveal much significant changes in stability and order. The linker loop reveals increase in order and stability in the mutated variants. Our studies may shade important light to the virulence of the viral species. The cryo EM structure of SARS CoV-2 spike trimer in a tightly closed state (7DF3) as well as in its open state (7DK3) are considered to study the conformational dynamics of spike protein and its effect on mutation. The mutations are obtained from the cryo EM structure. We have considered the most common mutations appeared in VOC strains like K417N, L452R, E484K and N501Y. We have also chosen a system which contains multiple mutations together designated as 'mt(mult)-spike' and a separate system which include only H69del and V70del without any other spike mutations as 'mt(del)-spike'. We perform 1 μ s-long all-atom MD simulation using the standard protocol for isothermal isobaric ensemble (NPT) with 310 K and 1-atm pressure in GROMACS 31 package. We use periodic boundary conditions, spc216 water model and GROMOS9353a6 32 force field for simulations in GROMACS 2018.6 package. Electro-neutrality is maintained by adding monovalent ions Na+ and Cl-. Long ranged columbic interactions are considered using PME approach 33 . LINCS algorithm 34 is used to constraint the bonds and leap-frog integration is used to perform simulation. Minimization is done for 50,000 steps using the steepest descent algorithms. Equations of motion are integrated using leap-frog algorithm with an integration time step of 2fs. Systems are equilibrated through 2 steps (NVT & NPT) using position restraints to heavy atoms. NVT and NPT equilibration is carried out at 300K Temperature and 1 Bar pressure. We maintain the total number of particles (N = 166251), pressure and temperature same for all the systems to make the simulated ensembles equivalent. are assessed from the saturation of the root mean squared deviations, RMSD. All the data have been averaged over six independent trajectories for each system. Conformational thermodynamics changes for spike proteins and its mutated varieties at different conformations are estimated properly from equilibrium fluctuations of the dihedral angles using the Histogram Based Method (HBM) 30 . Equilibrium conformational changes in free energy is sum is taken over histogram bins. In the current study we have focused only into the S1 subunit that interacts with the host cell receptor ACE2, consisting of the NTD (14-306 residues), the RBD (331-528 residues)and To quantify the conformational changes, we consider the centers of mass for NTD and RBD region respectively from the equilibrated trajectory of each of the system and then the distance S between the centers of mass of NTD and the RBD arm has been calculated over The flexibility of the protein conformations is given in terms of the dihedral fluctuations. Let us first consider the critical residues of the RBD region playing vital role in ACE2 interaction. We observe that ‫ܪ‬ . 3a) , while in rest of the mutated system, increase in flexibility can be found in his degree of freedom due to mutation. The same trend can be found in the backbone dihedral distribution (ψ) of this residue. 3b ). In this N487, huge increase in flexibility is noticed due to the side chain (χ 1 ). The sharp peak of ‫ܪ‬ changes into multimodal peaks due to mutations ( fig. 3c) . does not show much difference due to mutation (fig. 3f ). The cases of the dihedral angles of the other residues forming interacting with host factor of the receptor binding motif are shown in SI figs S1-S6. Overall, the distribution of dihedral angles shows an increase in flexibility in the RBD region due to spike protein mutation. Let us now consider the dihedral angle distributions of certain residues in the NTD domain. It is noticed that the dihedral distribution ߮ of Y170 is similar for all the system (fig. 4a) . show a sharp peak than the other systems which depicts enhanced flexibility due to mutation ( fig. 4b) . are sharp unimodal, whereas show bimodal distribution ( fig. 4c ), suggesting increase in flexibility due to such mutations. The cases of the dihedral angles of certain other residues from the NTD domain are shown in SI fig. S7 Now we consider residues from the linker loop. It is observed that show increase in flexibility ( fig. 5a) . Thus, mutation causes decrease in flexibility in this degree of freedom. Similarly, for ߰ of K310, decrease in flexibility is observed contributions from the residues of the particular region. We observe that (Table 1) NTD becomes energetically destabilized and disordered in the mutant variants than wt-spike system. However, NTD from the mt(del)-spike remain stabilized and more ordered with respect to wt-spike. The instability and disorder in the NTD arm are primarily dominated by backbone fluctuations (table 1 ). In mt(del)-spike, the side chain dihedral imparts maximum stability and ordering. The RBD residues of the spike protein undergo disorder and destabilization in the mutated system compared to the wt-spike system ( Table 2) . We observe that the mt(del)-spike system remains energetically stabilized and ordered with reference to wt-spike system. This trend is similar as found for NTD arm of the different systems of spike protein. The major contributions in free energy and entropy changes of the RBD region come from backbone dihedrals. It has been found that the overall entropy and free energy changes of the linker loop remains marginal (Table 3) , although the residues show more order and stabilization in the mutated case. This suggests that the linker residues play a role like a hinge to control the opening between RBD and NTD. It may be noted that the mt(del)-spike system, the linker shows disorder and destabilization where no hinge role is needed. Both the residue-wise and the domain wise free energy and entropy costs for conformational changes in the mutated protein with respect to the wt-type protein in the RBD and the linker regions are shown in SI Tables S1-S5. The overall change in entropy in the residues of RBD of mt(K417N)-spike is 5.62 KJ/mol where the major changes are in the backbone dihedrals. The total change in free energy is 7.28 KJ/mol in which the backbone dihedrals contribute the most (SI Table S1 ). In case of another point mutation mt(L452R)-spike, the total change in entropy of the region is 11.86 KJ/mol, where the backbone dihedrals together account for most of the changes (SI Table S2 ). The changes in the stability are marginal in all cases except Y449 having the highest change in stability. The total change in free energy is 4.8 Table S2 ). Altogether change in entropy of mt(E484K)-spike is 1.63 KJ/mol where the backbone dihedrals are the primary contributor. The backbone dihedral ሺ ߮ ሻ is the key factor for changes in free energy of mt(E484K)-spike in this region, the overall change of free energy being 1.66 KJ/mol (SI Table S3 ). The total changes in entropy of the region for mt(N501Y)spike is 8.59 KJ/mol, which is exhibited by the total changes in the backbone dihedrals (SI Table S4 ). The change in free energy is 5.35 KJ/mol where ሺ ߮ ሻ backbone dihedral acts as pivotal for the changes (SI Table S4 ).The backbone dihedrals contribute for major changes of the region, the total changes in entropy and free energy in mt(mult)-spike being 9.09 KJ/mol and 6.71 KJ/mol respectively (SI Table S5 ).The overall changes in entropy and free energy in the mt(del)-spike is changes in the system (SI Table S6 ). The details of conformational thermodynamics data for the certain residues of NTD domain are given in SI Table S7- Table S13 ). The backbone dihedral distribution accounts for maximum changes in this system. In mt(L452R)spike the entropy change and free energy change is -9.85 KJ/mol and -4.99 KJ/mol respectively (SI Table S14 ). Such ordering and stability in free energy is governed by backbone dihedral fluctuations (SI Table S14 ). The total changes in entropy for K310, G311, Y313, F329 and P330 in mt(E484K)-spike is -6.81 KJ/mol, primarily due to the backbone dihedral distributions. Besides, the total changes in free energy are -3.23 KJ/mol due to the backbone fluctuations. We find that P330 imparts slight increase of entropy (SI Table S15 ). In mt(N501Y)-spike, -6.92 KJ/mol is the total change in entropy and the total change in free energy is -4.7 KJ/mol (SI Table S16 ). However, the changes in entropy and free energy found due to such residues of linker loop in mt(mult)-spike is marginal, -0.53 KJ/mol and -1.87 KJ/mol respectively (SI Table S17 ). The mt(del)-spike system shows that the total change in entropy is 14.24 KJ/mol and the change in free energy is 3.47 KJ/mol (SI Table S18 ). We map the changes in conformational free energy G i conf and entropy T S i conf of individual residues of the mutated systems with respect to wt-spike. Here, we show free energetically stabilized and ordered residues in green and the destabilized and disordered ones in red. Careful examination of the ACE2 interacting residues of RBD of mt(K417N)-spike shows G446, Y449, N487, Y489, T500 and Y505 impart enhanced disorder, the maximum being in Y489. Increase in order is observed in Q493 and G502 (Fig. 6a) . It is found that Y449 and Y505 confers major decrease in stability, whereas, in rest of them the free energy change is marginal. In case of another point mutation mt(L452R)-spike, most of the interface residues which forms crucial interaction with host factor ACE2 gets disordered (Fig. 6b) . G446 and Y449 undergo maximum decrease in order. Q493 shows marginal increase in order. Y449 shows maximum destabilization in free energy of the region of mt(L452R)-spike. G446, N487, Y489, Q493, T500 and Y505 of mt(L452R)-spike show slight decrease in stability due to free energy change. G502 of mt(L452R)-spike account for minor increase in stability. The RBD residues of spike protein of mt(E484K)-spike show enhanced disorder in G446, N487, Y489 and T500, Y489 having the maximum disorder. On the other hand, Q493, G502, Y505 exhibit increase in order ( fig. 6c ). It has been found that Y449, N487, Y489 and T500 is responsible for decrease in stability in mt(E484K)-spike, the highest destabilized residue isY449. On the other hand, G446, Q493, To study the effect of the mutations on ACE2 recognition, we dock the native spike protein wt-spike and each of the mutated spike protein mt(del)-spike with ACE2 where the disordered and destabilized residues are taken to be active residues 30 In conclusion, we have performed detailed in-silico analysis of stability and order of the spike protein of SARS-CoV-2, primarily responsible for interaction with human cell receptor ACE2 . We find open conformation of the mutated protein, while a closed conformation of the wild type protein. 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