Journal of Biomolecular Structure and Dynamics

BackgroundSafety and efficacy of the SARS-CoV-2 inactivated vaccines have been question since the emergence of SARS-CoV-2 variants of concern (VOCs). Using residue fluctuations and statistically comparing RMSF values, have escalated the understanding of the binding dynamics of the viral proteins to their receptors and here in this study, we compared the interaction between inactivated spike proteins (representing FAKHRAVAC and BBIBP-CorV vaccines seed) and the human Angiotensin-Converting Enzyme 2 (hACE2) receptor. MethodologyThrough 100 set of accelerated 1 ns comparative molecular dynamics simulations, we analyze the binding dynamics and energy components of these interactions and compared residue backbone fluctuations using entropy and statistics including KL-Divergence and KS-test. Principal FindingsOur results reveal that FAKHRAVAC and Sinopharm exhibit similar binding dynamics and affinity to hACE2. Further examination of residue-wise fluctuations highlights the common behavior of binding key residues and mutation sites between the two vaccines. However, subtle differences in residue fluctuations, especially at critical sites like Q24, Y435, L455, S477, Y505, and F486, raise the possibility of distinct efficacy profiles. ConclusionThese variations may influence vaccine immunogenicity and safety in response to evolving SARS-CoV-2 variants. The study underscores the importance of considering residue-wise fluctuations for understanding vaccine-pathogen interactions and their implications for vaccine design. Author summaryIt is fundamentally important to ensure the safety and efficacy of the FAKHRAVAC, as an inactivated vaccine candidate for SARS-CoV-2. Considering the previously published pre-clinical and clinical findings about the similarity of the FAKHRAVACs safety and efficacy in comparison to the BBIBP-CorV vaccine seed (which is recalled as Sinopharm), it is necessary to gain more insights into structure and function of this vaccine at the molecular level, as well. Since the binding dynamics of the viral proteins to their receptor can imply the vaccines immunogenicity and mechanism-of-action, binding dynamics of a vaccine candidate must be studied comprehensively. Hereby, we have compared binding dynamics of the FAKHRAVAC and Sinopharm vaccine seeds to the SARS-CoV-2 spike proteins receptor, the ACE2. We took advantage of a comparative molecular dynamics simulation approach to effectively compare binding dynamics using atom fluctuations and at the residue level to ensure the resolution of this study. We have found similar binding dynamics and binding mechanics between these two vaccines, validating the pre-clinical and clinical findings computationally, as well as highlighting residues with different fluctuations and discussed their potential roles.

The Polo Like Kinases including the major family member, PLK1 are key regulatory enzymes controlling the cell cycle and mitosis. PLK1 is associated with poor survival rates in cancer and has been extensively investigated as an oncology drug target. Each member of the Polo like kinase family (PLKs 1-5) have two subdomains with independent functions and include the well conserved N-terminal kinase domain (KD) and the C-terminal polobox domain (PBD). The PBD is involved in the recognition of substrates primed by other kinases and in the PLK1 context is responsible for subcellular localization to specific sites in the nucleus including centrosomes and kinetochores. While the phosphosubstrate recognition site in PLKs 1-3 is highly conserved, its role in PLKs 2 and 3 is not well characterized and phosphopeptides that inhibit PLK1 have dramatically lower affinity for PLKs 2 and 3. An additional role of the PBD is its ability through domain-domain interactions with the KD to regulate PLK1 activity by an autoinhibited state of PLK1, conceptually similar to that which occurs through other kinases. Other mechanisms regulating PLK activity include the interchange between monomeric and dimeric forms, which inhibit or activate PLK1 during the cell cycle. Furthermore, PLK1 may exist as heterodimers with PLK2 and/or PLK3 and thus play context dependent roles. Here, through the use of the AlphaFold (AF) algorithm, structural insights into regulation of activity of the PLK1 and other family members have been obtained. These include dramatically different tertiary arrangement of the individual domains in each individual PLK. Analysis of the domain-domain interactions, interdomain and intradomain loops in each PLK sheds light onto plausible mechanisms by which the activity of each PLK is regulated and provides insights into the selectivity of phosphopeptides. The results also suggest a mechanism for the heterodimerization of PLK1 and PLK2 which has been observed in the literature.

Signal Transducer and Activator of Transcription 3 (STAT3) plays a crucial role in cancer development and thus is a viable target for cancer treatment. STAT3 functions as a dimer mediated by phosphorylation of the SRC-homology 2 (SH2) domain, a key target for therapeutic drugs. While great efforts have been employed towards the development of compounds that directly target the SH2 domain, no compound has yet been approved by the FDA due to a lack of specificity and pharmacologic efficacy. Studies have shown that allosteric regulation of SH2 via the coiled-coil domain (CCD) is an alternative drug design strategy. Several CCD effectors have been shown to modulate SH2 binding and affinity, and at the time of writing at least one drug candidate has entered phase I clinical trials. However, the mechanism for SH2 regulation via CCD is poorly understood. Here, we investigate structural and dynamic features of STAT3 and compare the wild type to the reduced function variant D170A in order to delineate mechanistic differences and propose allosteric pathways. Molecular dynamics simulations were employed to explore conformational space of STAT3 and the variant, followed by structural, conformation, and dynamic analysis. The trajectories explored show distinctive conformational changes in the SH2 domain for the D170A variant, indicating long range allosteric effects. Multiple analyses provide evidence for long range communication pathways between the two STAT3 domains, which seem to be mediated by a rigid core which connects the CCD and SH2 domains via the linker domain (LD) and transmits conformational changes through a network of short-range interactions. The proposed allosteric mechanism provides new insight into the understanding of intramolecular signaling in STAT3 and potential pharmaceutical control of STAT3 specificity and activity. Author SummaryIn all living organisms, the proliferation and survival of cells are regulated by various proteins. Signal Transducers and Activators of Transcription 3(STAT3) protein is one of the important proteins. However, the abnormal regulation of these proteins will lead to cancer cell. The constitutive activation of STAT3 has been linked to several types of solid tumors, leukemia, and lymphomas. Consequently, STAT3 proteins have been a key target for cancer therapy. SH2(SRC-homology 2) domain is the key interaction site, great efforts have been attributed to target SH2 domain, which specificity has been a major challenge in drug discovery. Research showing regulation of SH2 domain via CCD has opened a new path for drug discovery, however is challenged by poor understanding of the allosteric mechanism. Here, we show that CCD regulates SH2 conformation via a rigid backbone. The perturbations in CCD is transmitted through -helix to the rigid core that concert the movement of CCD and LD (Link domain), leading to structural changes in the SH2 domain. The present findings provide allosteric mechanism with atomistic details underlying the regulation of CCD to SH2 domain in STAT3 protein. Which allows informed drug design targeting CCD for desired downstream effect on SH2 domain and the overall STAT3 function.

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 bifunctional methylenetetrahydrofolate dehydrogenase/cyclohydrolase (MTHFD2) has been recognized as a promising anticancer drug target because it is overexpressed in various types of cancer and is associated with poor prognosis. In the present study, we aimed to discover potential inhibitors from the Enamine HTS library which consists of over one million compounds. A consensus docking-based virtual screening workflow was adopted and two hits, E96 and E41, were identified for being ranked in the top 5 in all docking programs used. To validate the virtual screening result, the binding modes of the two hits were visually inspected with reference to previously published inhibitors B01 and D56, and a similar pattern of binding was observed between the hits and established ligands, indicating the reliability of the docking protocol. The subsequent molecular dynamics simulation and a series of analyses including root mean square deviation, root mean square fluctuation, and radius of gyration reveal that E96 achieved a more stable binding to the receptor than E41. The binding free energy predicted by MM/GBSA calculation confirms E96s potential to be a potent inhibitor for the target MTHFD2 as it outperforms E41 and the established ligands. In conclusion, this computational study contributes to the drug discovery efforts for the anticancer drug target MTHFD2 by suggesting ligand E96 for further structure-based optimization and in vitro/vivo experimental validation.

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.

Prevailing COVID-19 vaccines are based on the spike protein of earlier SARS-CoV-2 strain that emerged in Wuhan, China. The continuously evolving nature of SARS-CoV-2 resulting emergence of new variants raises the risk of immune absconds. During the last few months, several RBD (receptor-binding domain) variants have been reported to affect the vaccine efficacy considerably. Soon after reporting of a new double mutant variant (L452R & E484Q) in India, the country facing a deadlier second wave of infections which prompts researchers to suspects this variant to be accountable. To address the relevant concerns about this new variant affecting vaccine efficacy, we performed molecular simulation dynamics based structural analysis of spike protein of double mutant (L452R & E484Q) along with K417G variants and earlier reported RBD variants and found structural changes in RBD region after comparing with the wild type. Comparison of the binding affinity of the double mutant and earlier reported RBD variant for ACE2 (angiotensin 2 altered enzymes) receptor and CR3022 antibody with the wild-type strain revealed the lowest binding affinity of the double mutant for CR3022 among all other variants. These findings suggest that the newly emerged double mutant could significantly reduce the impact of the current vaccine which threatens the protective efficacy of current vaccine therapy.

The causative agent of gastroenteritis is Shiga toxin, which belongs to a functionally and structurally associated protein family despite each individual having a unique amino acid sequence. After entering the ER lumen and relocating the toxic domain to the cytoplasm, they alter the large subunit of rRNA, preventing protein synthesis and ribosomal damage. Shiga-like toxin-1 (SLT-1) subunit B targets glycolipid receptor Gb3, which plays a significant role in cytotoxicity. Though the mutational effect on subunit B is important for cytotoxicity study, we lack better understanding. Our present study targets the mutational impact of glycine protein at their 62th amino acid sequence of subunit B. For example, how it can alter the receptor-binding capacity and virulence. We used in silico method with GROMACS software suite (version 5.2, 2020.1) on Google Colab for a 100ns (100,000ps) simulation period and UCSF Chimera software for visualizing mutant and wild-type structure similarities. Surprisingly, RMSD, RMSF, and Rg trajectories from the simulation analysis indicated a more stable and compact mutant structure than the wild type. Principle component analysis (PCA) and SASA were visualized for the entire 100ns, which pointed towards homogeneity between both structures and more solvent accessibility in the mutant structure. This mutation may elevate receptor-binding and virulence capacity. Moreover, this finding can offer a better insight for future vaccine production.

BackgorundRhoB is a key member of the Rho family of isoprenylated small GTPases which modulate the cellular cytoskeletal organization. It has a crucial role in the neoplastic apoptotic mechanism after DNA damage. Due to the unavailability of 3D structure in the protein data bank database, in this study, we evaluated the structure of a protein, Rho-related GTP-binding protein RhoB. ResultsRhoB has a predicted pI of 5.10, indicating that it is acidic. The GMQE value was used to compute the target-template alignment, and 6hxu.1.A from Homo sapiens was chosen as the template structure, with the model construction task completed using swiss-model. The structural compactibility and stability were revealed after a 100ns molecular dynamics simulation using GROMACA employing the OPLS-AA force field. PCA analysis found residues that are relevant based on their fluctuation acitivity while their location is between 100-110 and 140-150. ConclusionThis study will benefit future investigations addressing the association between gene mutation and abnormalities generated by protein Rho-related GTP-binding protein RhoB in apoptotic events by offering insight into the biophysical phenomenon of Rho-related GTP-binding protein RhoB inhibitors.

Dengue NS2B-NS3 protease existing in equilibrium between the active and inactive forms is essential for virus replication, thus representing a key drug target. Here Myricetin, a plant flavonoid, was characterized to non-competitively inhibit Dengue protease. Further NMR study identified the protease residues perturbed by binding to Myricetin, which were utilized to construct the Myricetin-protease complexes. Strikingly, in the active form Myricetin binds a new allosteric site (AS2) far away from the active site pocket and allosteric site (AS1) for binding Curcumin, while in the inactive form it binds both AS1 and AS2. To decipher the mechanism for the allosteric inhibition by Myricetin, we conducted molecular dynamics (MD) simulations on different forms of Dengue NS2B-NS3 protease. Unexpectedly, the binding of Myricetin to AS2 is sufficient to disrupt the active conformation by displacing the characteristic NS2B C-terminal {beta}- hairpin from the active site pocket. By contrast, the binding of Myricetin to AS1 and AS2 results in locking the inactive conformation. Therefore Myricetin represents the first small molecule which allosterically inhibits Dengue protease by both disrupting the active conformation and locking the inactive conformation. The results enforce the notion that a global allosteric network exists in Dengue NS2B-NS3 protease, which is susceptible to allosteric inhibition by small molecules such as Myricetin and Curcumin. As Myricetin has been extensively used as a food additive, it might be directly utilized to fight the Dengue infections and as a promising starting for further design of potent allosteric inhibitors. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=117 SRC="FIGDIR/small/472523v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@ec52d1org.highwire.dtl.DTLVardef@1313dc4org.highwire.dtl.DTLVardef@1ce40b5org.highwire.dtl.DTLVardef@1fa6051_HPS_FORMAT_FIGEXP M_FIG C_FIG

Despite improved treatment options, colorectal cancer (CRC) remains a huge public health concern with a significant impact on affected individuals. Cell cycle dysregulation and overexpression of certain regulators and checkpoint activators are important recurring events in the progression of cancer. Cyclin-dependent kinase 1 (CDK1), a key regulator of the cell cycle component central to the uncontrolled proliferation of malignant cells, has been reportedly implicated in CRC. This study aimed to identify CDK1 inhibitors with potential for clinical drug research in CRC. Ten thousand (10,000) naturally occurring compounds were evaluated for their inhibitory efficacies against CDK1 through molecular docking studies. The stability of the lead compounds in complex with CDK1 was evaluated using molecular dynamics simulation for one thousand (1,000) nanoseconds. The top-scoring candidates ADME characteristics and drug-likeness were profiled using SwissADME. Four hit compounds namely spiraeoside, robinetin, 6-hydroxyluteolin, and quercetagetin were identified from molecular docking analysis to possess the least binding scores. Molecular dynamics simulation revealed that robinetin and 6-hydroxyluteolin complexes were stable within the binding pocket of the CDK1 protein. The findings from this study provide insight into novel candidates with specific inhibitory CDK1 activities that can be further investigated through animal testing, clinical trials, and drug development research for CRC treatment.

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.

Hexanucleotide repeat expansions (HRE), located in the first intron of chromosome 9 open reading frame 72 (C9orf72) are the most common genetic abnormality associated with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Presence of the HRE may cause various effects to neuronal cells, leading to pathogenicity. One of these is the sequestration of RNA-binding proteins by three-quartet parallel RNA G-quadruplexes (RG4s) formed from repeated (GGGGCC)n sequences on the sense transcripts of the HRE. Multiple studies imply a major role of the sequestration of heterogeneous nuclear ribonucleoprotein H (hnRNP H) in the pathology of ALS/FTD. In this study, molecular docking and molecular dynamics (MD) were used to simulate the interaction of the three RNA recognition motifs (RRMs) of hnRNP H with the RG4. Molecular Mechanics with Generalised Born and Surface Area Solvation (MM-GBSA) and hydrogen bonding analyses of MD simulations were performed. The MM-GBSA analyses revealed that Arg29, Arg150, and Arg299 are important contributors to the binding, consistent with previous observations of arginine-mediated binding of protein to RNA. In addition, our results point to a previously unknown role of the stretch of residues from Lys72 to Tyr82 on hnRNP H for binding the (GGGGCC)n RG4, forming a hydrogen bonding hotspot. Interestingly, the identified residues are not located in the beta sheet, as would be expected of RRMs in general, suggesting that the binding of hnRNP H to this pathological RG4 may be specifically targeted. This has implications for future in vitro studies including but not limited to mutational analysis of these mentioned residues as well as drug development to prevent the sequestration of hnRNP H in ALS/FTD.

The PDZ domain is a highly abundant protein-protein interaction domain that exists in many signaling proteins, such as PICK1. Despite the highly conserved structure of the PDZ family, the PDZ family has an extremely low sequence identity, making each PDZ domain unique. PICK1 is the only protein in the human genome that is comprised of a PDZ domain and a BAR domain. PICK1 regulates surface membrane proteins and has been identified as an integral player in drug addiction. Like many PDZ-containing proteins, PICK1 is positively regulated by its PDZ domain and has thus drawn attention to be a potential drug target to curb the effects of substance abuse. The goal of this study is to use all-atom molecular dynamics simulations and the electrostatic analysis program, DelPhi, to better understand the unique interactions and dynamic changes in the PICK1 PDZ domain upon complex formation. Our results demonstrated that the PICK1 PDZ domain shares similar canonical PDZ-ligand hydrogen bonding networks and fluctuations of the carboxylate-binding loop to other PDZ domains. Furthermore, our results are unique to the PICK1 PDZ domain as we reveal that the binding of ligand opens up the binding pocket and, at the same time, reduces the fluctuations of both the central part of the binding pocket and the short loop region between the A-helix and {beta}C-strand. More importantly, the binding of ligand resulted in charge redistribution at the binding pocket region as well as the N- and C-termini of the PDZ domain that are not a part of the binding pocket. These results suggest that the electrostatic allostery resulted from ligand binding could be the key factor leading to the changes in dynamics which may be associated with the activation of PICK1. Based on these results, an effective drug to target PDZ domain must not only stably bind to the PICK1 PDZ domain but also prevent the electrostatic allostery of the PDZ domain.

The nucleotide-binding, leucine rich repeat, and pyrin-containing 3 (NLRP3) protein is regulated by phosphorylation of Ser295 in the NACHT domain. This post-translational modification is known to inhibit the enzymatic ATPase activity of NLRP3 and impede inflammasome complex assembly. In this study, modeled structures of unphosphorylated and pSer295-phosphorylated NLRP3-{Delta}PYD were subjected to molecular dynamics simulations. The outputs showed Ser295 phosphorylation to induce topologic distention of subdomains that comprise the NACHT domain. The relative orientation of important residues within the nucleotide-binding domain (NBD) were altered. Notable structural changes were observed for important residues within the Walker B motif that immediately follow pSer295. A favorable electrostatic environment was created for two residues (Lys232 and His522) that interact with ADP. Several other basic residues could establish favourable charge-charge interactions with the dianionic phosphate of pSer295. Arg296 and Glu343 underwent a functional change from negative/stabilizing to positive/destabilizing interaction upon phosphorylation of Ser295. Taken together, the results suggest that local structural transformations within the NBD could have consequences on the catalytic efficiency of the enzyme and suppress nucleotide turnover.

Scaffold proteins play pivotal role as modulators of cellular processes by operating as multipurpose conformation clamps. 14-3-3 proteins are gold-standard scaffold modules that recognize phosphoSer/Thr (pS/pT) containing conserved motifs of target proteins and confer conformational changes leading to modulation of their functional parameters. Modulation in functional activity of kinases has been attributed to their interaction with 14-3-3 proteins. Herein, we have characterized Plasmodium falciparum 14-3-3 and its interaction with key kinase of the parasite, Calcium-Dependent Protein Kinase 1 (CDPK1) by performing various analytical biochemistry and biophysical assays. Towards this, we annotated PF3D7_0818200 as 14-3-3 isoform I through extensive phylogenetic and comparative sequence analysis. Molecular dynamics simulation studies indicated that phosphoSer64 present in CDPK1 polypeptide sequence (61KLGpS64) behaves as canonical Mode I-type (RXXpS/pT) consensus 14-3-3 binding motif, mediating the interaction. The protein-protein interaction was validated in vitro with ELISA and SPR, which confirmed that CDPK1 interacts with 14-3-3I in a phosphorylation dependent manner, with binding affinity constant of 670 {+/-} 3.6 nM. The interaction of 14-3-3I with CDPK1 was validated with well characterized optimal 14-3-3 recognition motifs: ARSHpSYPA and RLYHpSLPA as CDPK1 mimetics, by simulation studies and ITC. Further, interaction antagonizing peptidomimetics showed growth inhibitory impact on the parasite indicating crucial physiological role of 14-3-3/CDPK1 interaction. Overall, this study characterizes 14-3-3I as a scaffold protein in the malaria parasite and unveils CDPK1 as its previously unidentified target. This sets a precedent for the rational design of 14-3-3 based PPI inhibitors by utilizing 14-3-3 recognition motif peptides, as a potential antimalarial strategy.

The formations of nucleoprotein structures by promiscuous DNA binding proteins like HU are assisted with their protein protein interaction capability with other proteins. In E. coli Gal repressosome assembly, GalR piggybacks HU to the critical position on the DNA (hbs site) through a specific GalR-HU interaction using an interface at the bottom of alpha helical region, which we termed as HUpb interface. Similarly, MtbHU also interact with Topoisomerase I with the same interface to enhance its relaxation activity. In an earlier study, we determined the crystal structure of MtbHU, inhibited it using stilbene derivatives which inhibited the cell growth. It motivated us to understand the evolutionary and structural characteristics of the HUpb interface, which has not been investigated previously for HU or for any other NAPs. Our analyses found residue positions corresponding to MtbHU Thr11 to Gln20 form the interface while Ala23 serves the pocket lining residue. Due to ancestral mutations in the duplication event before the HU and IHF split, physicochemical properties of the interface vary among clades. Thus, this interface could engage different proteins in different HU oligomeric states in Proteobacteria. Protein-protein interfaces are usually a challenging target due to its flatter surface. In case of MtbHUpb interface, we observed that due to the presence of a partially hydrophobic pocket, small molecule scaffolds could fit into it, while the ligand can be further designed to interact with D17, which is the crucial residue for Topoisomerase I interaction. We used a two-step virtual screening protocol with known drug like molecules as starting set to an aim to re-purpose drugs. Our docking results showed compounds like Maltotetraose, Valrubicin, Iodixanol, Enalkiren, indinavir, Carfilzomib, oxytetracycline, quinalizarin, Raltitrexed, Epigallocatechin and their analogues exhibit high scoring binding at MtbHUpb interface. Our present report gives a model example of an evolutionary study of an interface of nucleoid associated protein, which is used to computationally design inhibitors. This strategy could be in general useful for designing inhibitors for various types of protein-protein interfaces using evolutionary guided design.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to mutate and evolve with the emergence of omicron (B.1.1.529) as the new variant of concern. The rapid spread of this variant regionally and globally could be an allusion to increased infectivity, transmissibility, and antibody resistance. The omicron variant has a large set of mutations in its spike protein, specifically in the receptor binding domain (RBD), reflecting their significance in ACE2 interaction and antibody recognition. We have carried out the present study to understand how these mutations structurally impact the binding of the antibodies to their target epitope. We have computationally evaluated the binding of different classes of RBD targeted antibodies, namely, CB6 (etesevimab), REGN10933 (casirivimab), S309 (sotrovimab), and S2X259 to the omicron mutation-induced RBD. Molecular dynamics simulations and binding free energy calculations unveil the binding affinity and stability of the antibody-RBD complexes. All the four antibodies show reduced binding affinity towards the omicron RBD. The therapeutic antibody CB6 aka etesevimab was substantially affected due to numerous omicron mutations occurring in its target epitope. This study provides a structural insight into the reduced efficacy of RBD targeting antibodies against the SARS-CoV-2 omicron variant.

The 70 kDa heat shock proteins from Plasmodium falciparum (PfHSP70) are an important family of proteins that may be exploited for antimalarial design. Plant derived inhibitor lapachol is reported to efficiently inhibit the exported PfHSP70-x while having poor activity against the parasite PfHSP70-1. In the present study, we have used in silico tools including molecular docking to understand the molecular basis for the above differences in lapachol inhibition activity against PfHSP70s. We found a significant gap in the binding energies and hence affinity of PfHSP70-1 and PfHSP70-x for lapachol with the latter having a better binding propensity. Our data highlight notable differences in the type of interactions between the two complexes. Detailed molecular analysis of the complexes has helped us to predict specific amino acid residues from both these PfHSP70 homologs that may be involved in lapachol binding. The above information may be utilized for design of PfHSP70 inhibitors with antimalarial potential.

Mutation of an invariant aspartate residue in the binding pocket of 14-3-3{zeta} isoform to alanine dramatically reduced phosphopeptide binding and induced opening of the binding pocket. Here we use extensive molecular dynamics simulations to understand the role of D124 residue in ligand binding. The simulations show that in the absence of phosphopeptide, the D124A mutation leads to binding pocket reorganization including widening up of the binding pocket at the major groove and repositioning of N173, a key residue that interacts with the main chain of phosphopeptide. These structural changes would interfere with the efficient binding of the peptide, corroborating the experimental observations. Both gain and loss of electrostatic interactions in the form of salt bridges strongly indicate a rearrangement of the network of interactions within the binding pocket. Limited proteolysis coupled mass spectrometry (lip-MS) of the apo and holo forms of WT and mutant protein shows a peptide binding helix otherwise buried in the WT protein was particularly accessible to trypsin in the apo form of the mutant protein and the region was mapped to 158-186 amino acid residues of 14-3-3{zeta}). These results further confirm the dynamic nature of D124A mutant. Unlike other basic residues, the invariant D124 facilitates peptide binding by maintaining the geometry of interacting residues and by enforcing the structural integrity of amphipathic pocket.