Journal of Molecular Graphics and Modelling

The machinery involved in cytotoxic T-cell activation requires three main characters such as: the major histocompatibility complex class I (MHC I) bound to the peptide (p), the T-cell receptor (TCR), and the CD3-complex which is a multidimer interfaced with the intracellular side. The pMHC:TCR interaction has been largely studied both in experimental and computational models, giving a contribution in understanding the complexity of the TCR triggering process. Nevertheless, a detailed study of the structural and dynamical characterization of the full complex (pMHC:TCR:CD3-complex) is still missing, due to insufficient data available on the CD3-chains arrangement around the TCR. The recent determination of the TCR:CD3-complex structure by means of Cryo-EM technique has given a chance to build the entire proteins system essential in the activation of T-cell, and thus in the adaptive immune response. Here, we present the first full model of the pMHC interacting with the TCR:CD3-complex, built in a lipid environment. To describe the conformational behaviour associated with the unbound and the bound states, all atoms Molecular Dynamics simulations were performed for the TCR:CD3-complex and for two pMHC:TCR:CD3-complex systems, bound to two different peptides. Our data point out that a conformational change affecting the TCR Constant {beta} (C{beta}) region occurs after the binding to the pMHC, revealing a key role of such a region in the propagation of the signal. Moreover, we found that the TCR reduces the flexibility of the MHC I binding groove, confirming our previous results.

The new variant of SARS-CoV-2, Omicron, has been quickly spreading in many countries worldwide. Compared to the original virus, Omicron is characterized by several mutations in its genomic region, including spike proteins receptor-binding domain (RBD). We have computationally investigated the interaction between RBD of both wild-type and omicron variants with hACE2 receptor using molecular dynamics and MM-GBSA based binding free energy calculations. The mode of the interaction between Omicrons RBD to the human ACE2 (hACE2) receptor is similar to the original SARS-CoV-2 RBD except for a few key differences. The binding free energy difference shows that the spike protein of Omicron has increased binding affinity for the hACE-2 receptor. The mutated residues in the RBD showed strong interactions with a few amino acid residues of the hACE2. More specifically, strong electrostatic interactions (salt bridges) and hydrogen bonding were observed between R493 and R498 residues of the Omicron RBD with D30/E35 and D38 residues of the hACE2, respectively. Other mutated amino acids in the Omicron RBD, e.g. S496 and H505, also exhibited hydrogen bonding with the hACE2 receptor. The pi-stacking interaction was also observed between tyrosine residues (RBD-Tyr501: hACE2-Tyr41) in the complex, which contributes majorly to binding free energies suggesting this as one of the key interactions stabilizing the complex formation. The structural insights of RBD:hACE2 complex, their binding mode information and residue wise contributions to binding free energy provide insight on the increased transmissibility of Omicron and pave the way to design and optimize novel antiviral agents.

The COVID-19 pandemic has caused more than 424 million infections and 5.9 million deaths so far. The vaccines used against SARS-COV-2 by now have been able to develop some neutralising antibodies in the vaccinated human population and slow down the infection rate. The effectiveness of the vaccines has been challenged by the emergence of the new strains with numerous mutations in the spike (S) protein of SARS-CoV-2. Since S protein is the major immunogenic protein of the virus and also contains Receptor Binding Domain (RBD) that interacts with the human Angiotensin-Converting Enzyme 2 (ACE2) receptors, any mutations in this region should affect the neutralisation potential of the antibodies leading to the immune evasion. Several variants of concern (VOC) of the virus have emerged so far. Among them, the most critical are Delta (B.1.617.2), and recently reported Omicron (B. 1.1.529) which have acquired a lot of mutations in the spike protein. We have mapped those mutations on the modelled RBD and evaluated the binding affinities of various human antibodies with it. Docking and molecular dynamics simulation studies have been used to explore the effect of the mutations on the structure of the RBD and the RBD-antibody interaction. The analysis shows that the mutations mostly at the interface of a nearby region lower the binding affinity of the antibody by ten to forty per cent, with a downfall in the number of interactions formed as a whole and therefore, it implies the generation of immune escape variants. Notable mutations and their effect was characterised by performing various analyses that explain the structural basis of antibody efficacy in Delta and a compromised neutralisation effect for the Omicron variant. Our results pave the way for robust vaccine design that can be effective for many variants. Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY SynopsisThe research study utilises comparative docking and MD simulations analyses to illustrate how mutations in delta and omicron variants affect the binding of antibodies to the spike receptor binding domain (RBD) of SARS CoV-2.

Background and objectivesThe presence of penicillin-binding protein 2a (PBP2a) is the cause of Methicillin-resistant Staphylococcus aureus (MRSA), which is important nosocomial pathogens worldwide and now are also of growing importance in community-acquired infection. The PBP2a resistance depends upon a supplementary peptidoglycan transpeptidase, which continues to function when normal PBPs have been inactivated by beta-lactam antibiotics. Analysis and quantitative study of the molecular interactions of PBP2a against {beta}-lactam antibiotics are required, as they support explaining and enhance understanding of the structure-activity relationship of antibiotic resistance. MethodsBioinformatics and computational methods have been highly effective tools for {beta}-lactams targeting PBPs to tackle the urgent threat of antimicrobial resistance. Regarding {beta}-lactam antibiotics targeting PBP2a and PBPs, we applied different docking programs to illustrate inhibition mode, MM/GBSA to estimate the binding free energies, and molecular dynamic simulation to validate and analyze the molecular interactions. ResultsBased on {beta}-lactam antibiotics targeting PBPs as covalent inhibitors, covalent docking was employed to provide explicit models of PBP2a against susceptible {beta}-lactam antibiotics. The simultaneous use of non-covalent docking enhances our comprehensive comprehension of the resistance of PBP 2a, which resulted from the lack of covalent linked to {beta}-lactam antibiotics. The selected antibiotics strongly interact with PBP2a, revealing the essential amino acid residues and binding affinity for inhibition. MD simulations were performed for the ligand-bound state of PBP-2a to explain their interaction and conformational changes. These findings are also strongly supported by root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF) and Hydrogen bond analysis of the protein-ligand complex. ConclusionsOur research offers extensive knowledge of the PBP2a-lactam interactions for the ability of known antibiotics to combat MRSA. The simulation results indicating stability and accuracy provide valuable insights for the advancement of pharmaceutical interventions against infectious diseases O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=136 SRC="FIGDIR/small/639213v1_ufig1.gif" ALT="Figure 1"> View larger version (88K): org.highwire.dtl.DTLVardef@1dc180forg.highwire.dtl.DTLVardef@afb81borg.highwire.dtl.DTLVardef@6014c4org.highwire.dtl.DTLVardef@1f35113_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure abstract:C_FLOATNO The interaction of Methicillin with PBP2a of MRSA. The protein is shown as a cartoon model, and the covalent binding of the ligand and serine active site is shown as a stick model. C_FIG

Staphylococcus aureus (S. aureus), often perceived as an extracellular pathogenic agent, exhibits a remarkable ability to penetrate host cells, including endothelial, epithelial, and osteoblastic cells, significantly contributing to the pathogenesis of infections. A significant pathway for this invasion appears to involve the bacteriums binding to the 5{beta}1 integrin via a fibronectin bridge, followed by phagocytosis. Additionally, S. aureus presents staphylococcal protein A, a cell wall protein that binds to the Fc and Fab regions of immunoglobulins, playing a crucial role in virulence and immune evasion, and can also bind directly to the gC1qR receptor on endothelial cells. Furthermore, vitamin C is recognized for its antimicrobial and immunomodulatory properties, offering potential for reducing the risk of infection. Considering the aforementioned elements, our study focused on exploring the potential effects of vitamin C on the interactions between S. aureus and endothelial cells. Thus, we particularly examined two aspects: on the one hand, interactions involving fibronectin-binding proteins (FnBPs) proteins and human 5{beta}1 integrin, and on the other hand, the interaction between vitamin C and the direct entry receptor (the globular heads of complement component 1q receptor [gC1qR/p33], also known as hyaluronic acid binding protein 1 [HABP1]). To achieve this, we utilized molecular modeling assays, primarily relying on molecular docking.

Nuclear receptors are a family of transcription factors that activate respective genes when bound to specific ligands. Certain ligands induce conformational changes in the receptor which helps them recruit co-activators. The conformational changes induced by these ligands conformationally transition inactive conformation into an active conformation by changing the orientation of the C-terminal helix (H12). Despite their immense physiological importance, very few questions have been solved about the kinetics and the molecular mechanism of this transition from the inactive to active conformation. In this study, we have used extensive unbiased atomistic molecular dynamics simulations of Progesterone receptor bound to a partial agonist asoprisnil to investigate these two questions. Two different crystal structures for this complex provide us with a unique opportunity to study the conformational transition at the molecular level. Apart from elucidating several important dynamical information from these simulations, we used Markov state modeling to calculate the rate of the transition between the inactive and active-like states. More importantly, we have also shown the importance of non-native interactions in this conformational transition, which were seen to be formed during the transition from inactive to active-like conformation but not present in the active conformation itself. Apart from contributing to our fundamental understanding about the structure and dynamics of nuclear receptor at the molecular level, this study might be able to contribute to the larger problem of protein-folding itself.

MotivationHVEM-LIGHT binding regulates the immune system response in various ways: it co-stimulates T cell proliferation; promotes B cell differentiation and secretion of immunoglobulins; and enhances dendritic cell maturation. Strong and prolonged stimulation of T cells to proliferate causes high levels of IFN-{gamma}, which leads to chronic inflammation and is the reason for various autoimmune diseases. Therefore, blocking HVEM-LIGHT interaction may be a way to cure these diseases and prevent adverse reaction in organ and tissue transplantation. ResultsIn this work, we designed 62 peptides based on the CRDs of the HVEM structure, differentiating in the number and combination of disulfide bonds present. Based on extensive all-atom MD simulations in state-of-the-art force fields, followed by MM-GBSA binding energy estimation, we selected the most promising CRD2 variants interacting with LIGHT. Several point mutations of these variants provided us with the most strongly binding moiety: the CRD2 with a single disulfide bond (C58-C73) and K54E substitution. This result was supprased only by the truncated variants of CRD2(39-73) with the same disulfide bond present. The binding mechanism was investigated by the use of steered MD simulations, which showed the increased binding affinity of the abovementioned variants, while experimental circular dichroism was used to determine their structural properties. Availability and ImplementationThree PDB models of the LIGHT inhibitors: PM0084527, PM0084528, and PM0084592. Contactpkrupa@ifpan.edu.pl Supplementary informationOnline supplementary data is available at: .

Molecular mimicry between pathogen-derived and self-peptides shown by MHC molecules is one of the critical mechanisms in the pathophysiology of autoimmune diseases. Numerous studied has been conducted in this field to identify sequence similarity, but evaluating structural and dynamic similarity, systematic computational frameworks remain limited. Therefore, we created an automated multi-parameter molecular dynamics analysis workflow and used it to compare three peptides (KP1, KP2, and KP3) generated from Klebsiella pneumoniae bound to HLA-B class protein with one human self-peptide (Annexin-derived, ANX). We assessed six complementing parameters using one microsecond-scale MD simulation: radius of gyration (Rg), solvent-accessible surface area (SASA), hydrogen bonding dynamics, MM-GBSA binding free energy, root mean square fluctuation (RMSF), and root mean square deviation (RMSD) to understand time-dependent structural and dynamic behaviour of all the peptide-HLA-B complex. Additionally, hydrogen bond occupancy and molecular mechanics generalised Born surface area (MM-GBSA) binding free energy calculations were performed to provide a more comprehensive assessment of complex stability. Our analysis suggests that KP1 exhibits structural features consistent with molecular mimicry, maintaining conformational stability, surface exposure, and interaction patterns comparable to ANX. In contrast, KP2 showed reduced stability, characterised by higher RMSD values and substantial hydrogen bond loss, whereas KP3 displayed intermediate behaviour, with relatively favourable energetics but noticeable conformational variability. Overall, the multi-parameter framework enabled differentiation among the candidate peptides based on combined structural, dynamic, and energetic properties. The workflow can be adapted for the analysis of larger peptide datasets and may provide a systematic approach for investigating potential autoimmune-relevant molecular mimics in microbial proteomes, with required adjustments according to the system.

1Members of the parvalbumin (PV) family of calcium binding proteins are found in a variety of vertebrates, where can they influence neural functions, muscle contraction and immune responses. It was reported that the -parvalbumin (PV)s AB domain comprising two -helices, dramatically increases the proteins calcium (Ca2+) affinity by {approx}10 kcal/mol. To understand the structural basis of this effect, we conducted all-atom molecular dynamics (MD) simulations of WT PV and truncated -parvalbumin ({Delta}PV) constructs. Additionally, we also examined the binding of magnesium (Mg2+) to these isoforms, which is much weaker than Ca2+ (Mg2+ actually does not bind to the {Delta}PV). Our key finding is that reorganization energies (RE) assessed using molecular mechanics generalized Born approximation (MM/GBSA) correctly rank-order the variants according to their published Ca2+ and Mg2+ affinities. The [Formula] of the {Delta}PV compared to the wild-type (WT) is 415.57{+/-}0.55 kcal/mol, indicating that forming a holo state of {Delta}PV in the presence of Ca2+ incurs a greater reorganization penalty than the WT. This is consistent with the {Delta}PV exhibiting lesser Ca2+ affinity than the WT ({approx}9.5 kcal/mol). Similar trend was observed for Mg2+ bound variants as well. Further, we screened for metrics such as oxygen coordination of EF hand residues with ions and found that the total oxygen coordination number (16 vs. 12 in WT:Ca2+ and {Delta}PV:Ca2+) correlate with the reported ion affinities (-22 vs. -12.6 kcal/mol in WT:Ca2+ and {Delta}PV:Ca2+), which indicates that AB domain is required for the protein to coordinate with maximal efficiency with the binding ions. To our surprise, no significant differences were observed between the Mg2+ bound WT and {Delta}PV isoforms. Additionally, we have screened for factors such as total number of waters, hydrogen bonds, protein helicity and {beta}-content for the entire protein, which enables us to understand the impact of lack of AB domain on the entire structure and not just binding sites. Our data indicate that AB improves the overall helicity ({approx}5%) in apo as well as holo forms. Particularly, AB increases -helicity in the D-helix residues (i.e., 60-65) upon ion binding by {approx}35% (90% vs. 55% in the Ca2+ bound WT and {Delta}PV, 60% vs. 20% in the Mg2+ bound WT and {Delta}PV), which likely contributes to high Ca2+ binding affinity. On the contrary, no significant effect on the overall {beta}-content was observed. Similarly, increased dehydration ({approx}50) and increase in total number of hydrogen bonds ({approx}7) were observed upon ion binding in both the WT and {Delta}PV systems, however, no significant differences were observed between the WT and {Delta}PV variants and also between Ca2+ and Mg2+ isoforms. We speculate that this is due to the partially folded apo state that was captured in our MD simulations, which might not be physiologically relevant as suggested by NMR experiments [1]. Also, we have identified seven different interactions that might play a key role in binding the AB domain with the CDEF helices, particularly the D22(AB)-S78(CDEF) hydrogen bond. Overall, this study indicates that local (i.e., the EF hands) as well as global factors play a role in improved ion binding due to AB domain.

Beta adrenergic receptors ({beta}ARs) are G protein-coupled receptors that control processes as varied as heart rhythm and vascular tone by binding agonists such as norepinephrine to induce downstream signaling pathways. Beta blockers antagonize {beta}ARs to downregulate their activity, thus reducing heart rate and lowering vascular tone. We developed new Rosetta structural modeling protocol to develop state-specific models of {beta}1AR, expressed in cardiac myocytes, as well as {beta}2AR, expressed in the smooth muscle cells of vasculature and other tissues, and their atomistic-scale interactions with beta-blockers using RosettaLigand. We identified structural features of drug - receptor interactions, which may account for their receptor conformational state and drug stereospecific preferences. Furthermore, we estimated structural stabilities of our models using atomistic molecular dynamics (MD) simulations. In our recent study we validated our structural models of norepinephrine-bound {beta}2AR and its complex with stimulatory G protein via multi-microsecond MD simulations. Thus, here we mostly focused on state-dependent and stereospecific {beta}1AR interactions with beta-blocking drugs sotalol and propranolol. We observed expected inactive receptor state preferences and structural stabilities of our models in MD simulations, but neither those simulations nor RosettaLigand docking could clearly distinguish stereospecific preferences of those drugs. This warrants consideration of alternative hypotheses and enhanced sampling MD simulations, which we discussed as well. Nevertheless, our study provides basis for understanding conformational state selectivity and stereospecificity of beta-blockers for {beta}ARs, important pharmacological targets, and may be extended to other drug classes and receptor types. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=157 SRC="FIGDIR/small/560334v1_fig0.gif" ALT="Figure 0"> View larger version (82K): org.highwire.dtl.DTLVardef@e8a838org.highwire.dtl.DTLVardef@7c2743org.highwire.dtl.DTLVardef@f5ac43org.highwire.dtl.DTLVardef@100a3d3_HPS_FORMAT_FIGEXP M_FIG Norepinephrine (NE) bound active-state beta-1 adrenergic receptor (1AR) in complex with the stimulatory G protein (Gs) heterotrimer embedded in a lipid bilayer. When expressed at the plasma membrane, the 1AR is oriented such that the ligand binding pocket (*) is accessible to ligands from the extracellular side (Ex.) of the membrane. The Gs (red), G (blue), and G{gamma} (yellow) subunits comprise the Gs heterotrimer. Nucleotides GDP or GTP bind G at the P-loop (**). Inset: Representative image of NE bound within the orthosteric ligand binding pocket. C_FIG

Remarkable infectivity of severe acute respiratory syndrome-coronavirus 2 (SARS-CoV2) is due to the rapid emergence of various strains, thus enable the virus to rule the world. Over the course of SARS-CoV2 pandemic, the scientific communities worldwide are responding to newly emerging genetic variants. However, the mechanism behind the persistent infection of these variants is still not known due to the paucity of study of these variants at molecular level. In this scenario, computational methods have immense utility in understanding the molecular and functional properties of different variants. Therefore, in this study various mutants (MTs) of SpikeS1 receptor binding domain (RBD) of highly infectious SARS-CoV2 strains were carried and elucidated the protein structure and dynamics using molecular dynamics (MD) approach. MD simulation study showed that all MTs exhibited stable structures with altered functional properties. Furthermore, the binding strength of different MTs along with WT (wildtype) was revealed through protein-protein docking and observed that MTs showed high binding affinities than WT. Hence, this study shed light on the molecular basis of infection caused by different variants of SARS-CoV2, which might play an important role in to cease the transmission and pathogenesis of virus and also implicate in rational designing of a specific drug.

The average human lifespan continues to increase with the increase in data flow and the advancement of related technological developments. However, this development brings with it many diseases, including immunological problems. Immunoglobulin varieties found in different organisms in the last 3-4 decades continue to be hope for many diseases. Interest has focused on the lesser weight but more mobile immunoglobulins found in camelids. Later, different types of these antibodies were tried to be made with biotechnological engineering and their effectiveness continues to be investigated. Disulfide bridges located on the immunoglobulin are one of the key points for the structure and function of the immunoglobulin. The interest of potassium hydroxide in disulfide bridges may enable us to damage or break these bonds. For this purpose, in this study, the relationship between disulfide bridges between light and heavy chains and potassium hydroxide was investigated. It was observed that the affinity of potassium hydroxide to disulfide bridges occurred exergonically. In the light of this information, it can be thought that lighter, more functional immunoglobulin fragments and nanobodies can be formed with potassium hydroxide compared to conventional immunoglobulin.

The outbreak of the 2019-nCoV coronavirus causing severe acute respiratory syndrome which can be fatal, especially in elderly population, has been declared a pandemic by the World Health Organization. Many biotechnology laboratories are rushing to develop therapeutic antibodies and antiviral drugs for treatment of this viral disease. The viral CoV spike (S) glycoprotein is one of the main targets for pharmacological intervention. Its receptor-binding domain (RBD) interacts with the human ACE2 receptor ensuring the entry of the viral genomes into the host cell. In this work, we report on the differences in the binding of the RBD of the previous coronavirus SARS-CoV and of the newer 2019-nCoV coronavirus to the human ACE2 receptor using atomistic molecular dynamics techniques. Our results show major mutations in the 2019-nCoV RBD with respect to the SARS-CoV RBD occurring at the interface of RBD-ACE2 complex. These mutations make the 2019-nCoV RBD protein backbone much more flexible, hydrophobic interactions are reduced and additional polar/charged residues appear at the interface. We observe that higher flexibility of the 2019-nCoV RBD with respect to the SARS-CoV RBD leads to a bigger binding interface between the 2019-nCoV RBD and ACE2 and to about 20% more contacts between them in comparison with SARS-CoV. Taken together, the 2019-nCoV RBD shows more stable binding interface and higher binding affinity for the ACE2 receptor. The mutations not only stabilize the binding interface, they also lead to overall more stable 2019-nCoV RBD protein structure, even far from the binding interface. Our results on the molecular differences in the binding between the two viruses can provide important inputs for development of appropriate antiviral treatments of the new viruses, addressing the necessity of ongoing pandemics.

AbstractGlycoprotein receptors are a subfamily of G-protein coupled receptors, including the follicle hormone (FSH) receptor (FSHR), thyroid-stimulating hormone receptor (TSH), and luteinizing/chorionic gonadotrophin hormone receptor (LHCGR). These receptors display common structural features such as a prominent extracellular domain, with a leucine-rich repeats (LRR) stabilized by {beta}-sheets, a long and flexible loop known as the hinge region (HR), and the transmembrane (TM) domain with seven -helices interconnected by intra- and extracellular loops. Binding of the ligand to the LRR resembles a hand coupling transversally to the - and {beta}-subunits of the hormone, with the thumb being the HR. The structure of the complex of FSHR-FSH suggests an activation mechanism in which Y335 at the HR binds into a pocket between the - and {beta}-chains of the hormone, leading to an adjustment of the extracellular loops. In this study, we performed molecular dynamics (MD) simulations to identify the conformational changes for the FSHR and LHCGR. We set up an FSHR structure as predicted by AlphaFold (AF-P23945); for the LHCGR structure we took the cryo-electron microscopy structure for the active state (PDB:7FII) as initial coordinates. Specifically, the flexibility of the HR domain and the correlated motions of the RLL and TMD were analyzed. From the conformational changes of the LRR, TMD, and HR we explored the conformational landscape by means of MD trajectories in all-atom approximation, including a membrane of polyunsaturated phospholipids. The distances and procedures here defined may be useful to propose reaction coordinates to describe diverse processes such as the active-to-inactive transition, to identify intermediaries suited for allosteric regulation, and biased binding to cellular transducers in a selective activation strategy. Author summaryIn the present study, we describe the results from a computational microscopy perspective (also known as molecular dynamics simulation) at the atomistic resolution for the two gonadotropin hormone receptors, the follicle-stimulant hormone receptor and the luteinizing/chorionic gonadotropin hormone receptor, which are essential for reproduction in humans. Several dysfunctional mutations in these receptors, leading to reproductive failure, have been detected in the clinical arena. To better understand the process whereby these two receptors perform their signaling tasks, triggering an intracellular response upon binding of their cognate agonist at the extracellular side, we assembled the receptor structures in a membrane bilayer of phospholipids with water molecules as solvent at both sides of the membrane. The systems included nearly 200 thousand atoms, each moving around at 300 kelvin and 1 bar given the interactions (attractive or repulsive forces) from each other. As the motion equations are solved in each time step (at femtoseconds time scale), the system evolves over time during hundreds of nanoseconds (millions of time steps) for three independent replicates. The receptor conformation, therefore, may display non-random motions due to the stability of specific structures in the complex molecular environment, including the hydrophobic membrane core, the bilayer interfaces, and the aqueous medium. From analysis of simulation trajectories and structural changes of the receptors, we could identify the main conformational changes exhibited by each receptor explored in a model cellular environment. We discussed the roll of the hinge domain at the extracellular domain in triggering the receptor conformational changes, as well as differences in the dynamics between these receptors in terms of the flexibility of the structures. Importantly, we proposed relative distances among the different receptor domains as parameters to characterize conformational intermediaries along a transition of states. Understanding of the signaling process in gonadotropin hormone receptors could be useful to explore new strategies for the modulation of the receptor functions, the bias of signaling pathways, or the selective binding of agonists.

Our study focuses on free energy calculations of SARS-CoV2 spike protein receptor binding motives (RBMs) from wild type and variants-of-concern with particular emphasis on currently emerging SARS- CoV2 omicron variants of concern (VOC). Our computational free energy analysis underlines the occurrence of positive selection processes that specify omicron host adaption and bring changes on the molecular level into context with clinically relevant observations. Our free energy calculations studies regarding the interaction of omicrons RBM with human ACE2 shows weaker binding to ACE2 than alphas, deltas, or wild types RBM. Thus, less virus is predicted to be generated in time per infected cell. Our mutant analyses predict with focus on omicron variants a reduced spike-protein binding to ACE2-receptor protein possibly enhancing viral fitness / transmissibility and resulting in a delayed induction of danger signals as trade-off. Finally, more virus is produced but less per cell accompanied with delayed Covid-19 immunogenicity and pathogenicity. Regarding the latter, more virus is assumed to be required to initiate inflammatory immune responses.

A recent fatal outbreak of novel coronavirus SARS-CoV-2, identified preliminary as a causative agent for series of unusual pneumonia cases in Wuhan city, China has infected more than 20 million individuals with more than 4 million mortalities. Since, the infection crossed geographical barriers, the WHO permanently named the causing disease as COVID-2019 by declaring it a pandemic situation. SARS-CoV-2 is an enveloped single-stranded RNA virus causing a wide range of pathological conditions from common cold symptoms to pneumonia and fatal severe respiratory syndrome. Genome sequencing of SARS-CoV-2 has revealed 96% identity to the bat coronavirus and 79.6% sequence identity to the previous SARS-CoV. The main protease (known as 3C-like proteinase/ Mpro) plays a vital role during the infection with the processing of replicase polyprotein thus offering an attractive target for therapeutic interventions. SARS-CoV and SARS-CoV-2 Mpro shares 97% sequence identity, with 12 variable residues but none of them present in the catalytic and substrate binding site. With the high level of sequence and structural similarity and absence of any drug/vaccine against SARS-CoV-2, drug repurposing against Mpro is an effective strategy to combat COVID-19. Here, we report a detailed comparison of SARS-CoV-2 Mpro with SARS-CoV Mpro using molecular dynamics simulations to assess the impact of 12 divergent residues on the molecular microenvironment of Mpro. A structural comparison and analysis is made on how these variable residues affects the intra-molecular interactions between key residues in the monomer and biologically active dimer form of Mpro. The present MD simulations study concluded the change in microenvironment of active-site residues at the entrance (T25, T26, M49 and Q189), near the catalytic region (F140, H163, H164, M165 and H172) and other residues in substrate binding site (V35T, N65S, K88R and N180K) due to 12 mutation incorporated in the SARS-CoV-2 Mpro. It is also evident that SARS-CoV-2 dimer is more stable and less flexible state compared to monomer which may be due to these variable residues, mainly F140, E166 and H172 which are involved in dimerization. This also warrants a need for inhibitor design considering the more stable dimer form. The mutation accumulated in SARS-CoV-2 Mpro indirectly reconfigures the key molecular networks around the active site conferring a potential change in SARS-CoV-2, thus posing a challenge in drug repurposing SARS drugs for COVID-19. The new networks and changes in microenvironment identified by our work might guide attempts needed for repurposing and identification of new Mpro inhibitors.

In India, the breakthrough infections during second wave of COVID-19 pandemic was due to SARS-COV-2 delta variant (B.1.617.2). It was reported that majority of the infections were caused by the delta variant and only 9.8% percent cases required hospitalization whereas, only 0.4% fatality was observed. Sudden dropdown in COVID-19 infections was observed within a short timeframe, suggesting better host adaptation with evolved delta variant. Down regulation of host immune response against SARS-CoV-2 by ORF8 induced MHC-I degradation has been reported earlier. The Delta variant carried mutations (deletion) at Asp119 and Phe120 amino acids which are critical for ORF8 dimerization. The deletions of amino acids Asp119 and Phe120 in ORF8 of delta variant results in structural instability of ORF8 dimer caused by disruption of hydrogen bonding and salt bridges as revealed by structural analysis and MD simulation studies of ORF8 dimer. Further, flexible docking of wild type and mutant ORF8 dimer revealed reduced interaction of mutant ORF8 dimer with MHC-I as compared to wild type ORF8 dimer with MHC-1, thus implicating its possible role in MHC-I expression and host immune response against SARS-CoV-2. We thus propose that mutant ORF8 may not hindering the MHC-I expression thereby resulting in better immune response against SARS-CoV-2 delta variant, which partly explains the sudden drop of SARS-CoV-2 infection rate in the second wave of SARS-CoV-2 predominated by delta variant in India Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/457457v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@751eeaorg.highwire.dtl.DTLVardef@140b5b5org.highwire.dtl.DTLVardef@159a3a5org.highwire.dtl.DTLVardef@6c206_HPS_FORMAT_FIGEXP M_FIG C_FIG

The influenza virus has caused a global pandemic with significant morbidity and mortality, highlighting the need to optimize antibodies for improved antiviral efficacy. The 3E1 antibody effectively neutralizes influenza subtypes H1 and H5 by inhibiting acid-induced conformational changes of hemagglutinin (HA). This study aimed to optimize the antibodys bioactivity by modifying amino acid residues, resulting in single-point mutants (3E1-L [W32I], 3E1-H [F103I]) and a double mutant (3E1-H+L [F103I, W32I]). The binding affinity, neutralizing activity, and antiviral mechanisms of the mutants were evaluated. Notably, the 3E1-L mutant showed significantly enhanced antiviral activity against H1N1 and H3N2 compared to wild-type 3E1, inhibiting both viral entry and release. The prophylactic and therapeutic efficacy of the 3E1-L mutant was validated. Molecular dynamics simulations of the 3E1-L/HA complex showed that the W32I mutation reduces steric hindrance between tryptophan at position 32 and the complementarity-determining region (CDR) L1 loop of HA. In conclusion, the W32I substitution enhances the antiviral activity of wild-type 3E1, making the optimization of 3E1-L a promising strategy for developing more effective influenza therapies.

CONSPECTUSO_ST_ABSAimsC_ST_ABSThe ribosomal protein (r-protein) of bacteria is composed of 2.6 MDa ribonucleoproteins of the 30S and 50S subunits, which are essential elements for protein translation. The translational initiation step is an intensive regulated multi-step reaction in protein biosynthesis. During bacterial protein synthesis, the correct reading frame of the mRNA defines when the initiator fMet-tRNAiMet binds to the start codon AUG at the P-site of the 30S subunit. The formation of the P-site of the 30S subunit initiation complex (30S-IC) is governed by three ubiquitous initiation factors (IFs) such as IF1, IF2, and IF3. IF2 protein is an essential player that plays during the last stage of the initiation process. Earlier, Stokes and his co-workers studied chemicals probes using 30K diverse drugs that induced cold-sensitive growth inhibition in the bacterium. The assay studies revealed, Lamotrigine (LTG) effectively binds at domain II of IF2 protein. In our research, we took an attempt in identifying promising active residues that could responsible for anti-bacterial bioactivity with help of computational studies. Computational MethodsIn the present study, initially, we performed C- backbone alignment with the retrieved IF2 chain from AlphaFold. Further, we utilized SiteMap and CastP for the identification of plausible active binding sites. Further, we bound LTG with the designated domain(s) of IF2 protein and studied its binding affinity potential with help of adaptive molecular dynamics simulations at atomic levels using Desmond. Key FindingsOur research findings have shown accurate results and we could able to prove the assertion in contrast with the findings of Stokes and his co-workers where the LTG bind at domain II of IF2 protein. The key interacting residue Glu179 was revealed to have strong hydrogen bonding contacts with LTG at the sub-nanomolar range. In addition, we predicted the alternative promising site I Further, we gained in-depth analysis for studying multiple sites, to understand the synergism inhibitory activity. Promisingly, LTG could be able to bound with at Site 1 showing better affinity over the proposed domain II and other predicted sites. The adaptive molecular dynamics studies confirmed the promising active residues SignificanceThe binding site predictions approach provides an insight for further development of anti-bacterial therapeutics that might helpful for bacteria disease management and exhibiting inhibitory activity against various strains.

The evolutionary and functional studies suggested that the emergence of the Omicron variants can be determined by multiple fitness trade-offs including the immune escape, binding affinity, conformational plasticity, protein stability and allosteric modulation. In this study, we embarked on a systematic comparative analysis of the conformational dynamics, electrostatics, protein stability and allostery in the different functional states of spike trimers for BA.1, BA.2, and BA.2.75 variants. Using efficient and accurate coarse-grained simulations and atomistic reconstruction of the ensembles, we examined conformational dynamics of the spike trimers that agrees with the recent functional studies, suggesting that BA.2.75 trimers are the most stable among these variants. A systematic mutational scanning of the inter-protomer interfaces in the spike trimers revealed a group of conserved structural stability hotspots that play a key role in modulation of functional dynamics and are also involved in the inter-protomer couplings through local contacts and interaction networks with the Omicron mutational sites. The results of mutational scanning provided evidence that BA.2.75 trimers are more stable than BA.2 and comparable in stability to BA.1 variant. Using dynamic network modeling of the S Omicron BA.1, BA.2 and BA.2.75 trimers we showed that the key network positions driving long-range signaling are associated with the major stability hotspots that are inter-connected along potential communication pathways, while sites of Omicron mutations may often correspond to weak spots of stability and allostery but are coupled to the major stability hotspots through interaction networks. The presented analysis of the BA.1, BA.2 and BA.2.75 trimers suggested that thermodynamic stability of BA.1 and BA.2.75 variants may be intimately linked with the residue interaction network organization that allows for a broad ensemble of allosteric communications in which signaling between structural stability hotspots may be modulated by the Omicron mutational sites. The findings provided plausible rationale for mechanisms in which Omicron mutations can evolve to balance thermodynamic stability and conformational adaptability in order to ensure proper tradeoff between stability, binding and immune escape.