Gaucher disease (GD) is a lysosomal storage disorder caused by deficient acid β-glucosidase (GCase) activity and consequent accumulation of glucosylceramide. Current enzyme replacement and substrate reduction therapies improve several systemic manifestations but have limited impact on neuronopathic disease, motivating evaluation of alternative molecular strategies. This study computationally assessed the interaction of EPA-phosphatidylcholine (EPA-PC), a marine-derived omega-3 phospholipid, with human GCase (PDB ID: 2NSX). The protein structure was examined by Ramachandran analysis and hydrophobicity profiling, followed by AutoDock Vina engine docking at the proposed hydrophobic i-face. EPA-PC showed a docking score of -5.5 kcal/mol under the applied protocol. Independent PLIP profiling of the docked complex identified predominantly hydrophobic contacts, one hydrogen bond involving Phe347, and Arg395-mediated salt-bridge interactions. A single 200 ns molecular dynamics trajectory of the EPA-PC-GCase complex showed an apparent backbone RMSD plateau of approximately 0.35-0.40 nm and a radius of gyration near 2.4 nm without a sustained increase in global compactness or solvent exposure. These observations support persistence of the modelled complex during the reported trajectory but do not establish ligand-induced GCase stabilisation or pharmacological-chaperone activity. In silico ADMET profiling also indicated substantial liabilities, including very high plasma protein binding and predicted hERG-related risk. EPA-PC should therefore be regarded as a preliminary lipid-protein interaction model requiring comparative simulations, independent replicates, and experimental validation.
WNTs are a family of signaling proteins involved in numerous biological processes, including morphogenesis, oncogenesis, cell migration and proliferation, cellular specialization, and tissue regeneration. WNT proteins are characterized by a distinctive structure comprising two domains resembling a “thumb” and an “index finger,” which enables their interaction with cellular receptors. Despite the identification of 19 WNT proteins in humans, three-dimensional structural data are available for only a subset, highlighting the importance of studying WNT proteins using in silico approaches. This study presents the in silico structural analysis of three human WNT family members — WNT1, WNT3A, and WNT5A — across multiple levels of protein organization. The primary structure of these proteins was analyzed for amino acid composition, and secondary structure predictions for α-helices, β-strands, and loops were correlated with domain structures characteristic of WNT proteins. Tertiary structures were modeled using homology modeling and deep-learning algorithms, and structural properties were investigated through molecular dynamics simulations. The ability of WNT proteins to adopt several closed and open conformational states was demonstrated, as well as the role of the flexibility of the β-hairpin forming the “index finger” in mediating conformational transitions. Thus, by applying a comprehensive bioinformatic approach to WNT proteins, the study highlights the advanced capabilities of in silico methods for analyzing protein structures at multiple levels of organization.
The enzymes Cannabichromenic Acid Synthase (CBCAS), Cannabidiolic Acid Synthase (CBDAS) and Tetrahydrocannabinolic Acid Synthase (THCAS) are together the major cannabinoid synthase enzymes responsible for the biosynthesis of their respective metabolites from a common precursor Cannabigerolic Acid (CBGA). As the catalysts responsible for generating biological molecules of significant pharmaceutical value, there has been considerable interest in the enzymes with respect to heterologous production, mechanism, and incorporation into synthetic biology pathways for the facile industrial production of these molecules. The enzymes share high degrees of homology, and therefore their distinct specificities are governed by very subtle differences in sequence and therefore structure, although, until now, only a structure for THCAS has been reported. In this report, we present structures of CBCAS, CBDAS and a structure of THCAS at a higher resolution than the known structure, each in complex with their flavin coenzyme FAD. The structures reveal active site differences that may be responsible for the complementary activities observed, in terms of both first-shell amino acid substitutions, but also in more remote residues that influence active site topology through referred effects, or that have effects on substrate access. The structures provide a useful and informative platform for the rational engineering of improved or altered chemoselectivity in these enzymes.
L-amino acids and nucleotide bases are essential biomolecules in all living organisms and represent attractive building blocks for designing safer, more effective drugs. In this study, we designed JAK kinase inhibitor candidates derived from these natural compounds and evaluated them using an integrated computational workflow. MarvinSketch was used to generate 100 structures, which were optimized in Spartan; frequency calculations confirmed the absence of imaginary modes and enabled calculation of key physicochemical properties. The candidates were then screened against five known JAK inhibitors using machine learning models (SVR, RF, KNN, and an ensemble approach), yielding (3S,5S)-5-((2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)methyl)-3-(4-aminobutyl)morpholin-2-one (AMO) as the top hit. Molecular docking identified favorable active-site interactions, and Abrocitinib (ABR) was selected as the closest comparator with the best overlay to AMO. Molecular dynamics simulations showed stable AMO binding, supported by RMSD/RMSF, radius of gyration, hydrogen-bonding, and DSSP analyses. PCA indicated a more compact conformational distribution for AMO than ABR, consistent with reduced large-scale motions, and DCCM supported favorable correlated dynamics. Pharmacokinetic profiling and ProTox-3.0 toxicity predictions classified both compounds as class 4, but AMO exhibited substantially lower predicted toxicity than ABR. Finally, MM-PBSA calculations yielded negative binding energies, confirming that the AMO-protein interaction is thermodynamically favorable and strong.
We have evaluated the quality of over 1200 crystal structures of hen egg-white lysozyme (HEWL) deposited in the Protein Data Bank (PDB). These structures, collected over nearly 50 years, vary in quality, despite all representing essentially the same small enzyme consisting of 129 amino acid residues. Some of the entries originated from studies of the binding of small-molecule ligands to HEWL, whereas the majority of deposits represent the outcomes of tests of new experimental approaches to crystallization and data collection and/or evaluations of new computational protocols. We found no correlation between Rfree, which is a measure of structure quality, and Rmerge, an indicator of raw data quality, for 136 near-atomic-resolution lysozyme structures. We found out that many of the lysozyme structures deposited as a result of methodology evaluation are not fully or correctly refined. We, therefore, propose that such structures be appropriately flagged in the PDB with a CAVEAT record to prevent their inadvertent inclusion in large-scale data mining analyses or training sets for artificial intelligence methods.
The RNA-binding protein hnRNPA2B1 is critical for mRNA processing, transport, metabolism, and antiviral innate immunity. Its activity is modulated by various ligands, including RNA, single-stranded DNA (ssDNA), and the small-molecule agonist PAC5, but the structural dynamics of these ligand-specific modulations are not fully understood. We hypothesized that each ligand triggers distinct conformational shifts that dictate functional outcomes. Starting from available crystal structures, we built three complex models and performed 100-ns molecular-dynamics simulations, analyzing RMSD, RMSF, radius of gyration, free-energy landscapes, MM/ PBSA binding affinities, PCA projections, and trajectory clustering. Our analyses reveal common and distinct interaction footprints between hnRNPA2B1 and the three ligands. Residues 24, 62, and 97 engage all ligands, whereas residues 28 and 30 form pronounced contacts with ssDNA yet only weakly interact with RNA and PAC5. Conversely, residues 102 and 108 are stably anchored to RNA but not to ssDNA. Radius-of-gyration and RMSF analyses further indicate that the backbone flexibility of hnRNPA2B1 is ligand-dependent: the protein is most expanded when bound to PAC5 and most compact when bound to ssDNA. Consistently, the binding free energy is highest for PAC5 and lowest for ssDNA. Finally, PCA shows that the PAC5-bound ensemble splits into two distinct conformational clusters whose overlap with the ssDNA-bound state (39.26 %) markedly exceeds that with the RNA-bound state (16.12 %), suggesting that PAC5 may promote hnRNPA2B1 dimerization by mimicking the ssDNA-bound conformation. These findings illuminate the ligand-dependent conformational and interaction landscapes of hnRNPA2B1, establishing a robust structure-thermodynamics framework for rational drug discovery.
RNase H enzymes are sequence-nonspecific endonucleases that cleave RNA strands in RNA/DNA hybrid duplexes, an enzymatic process essential in DNA replication and repair in both prokaryotes and eukaryotes. Also, RNase H activity of the reverse transcriptase in human immunodeficiency viruses (HIV-1 and HIV-2) is indispensable for the viral replication cycle. RNase H enzymes play an central role in the development of gene therapies and are targets for novel antivirals. It is therefore of great importance to gain a detailed understanding of the RNase H catalytic mechanism to improve drug design. We utilized Bacillus halodurans RNase H1 (BhRNase H1) to shed light on its function and catalytic mechanism. Room-temperature neutron crystallography of the wild-type and inactive D132N mutant enzymes revealed that E109, belonging to the catalytic DEDD motif, can change its protonation state, allowing us to propose its role in the protonation of the leaving O3 ' hydroxyl group of RNA. X-ray crystallography has demonstrated the ability of the RNA/DNA duplex to slide along the protein surface upon metal ion binding at site MA, transforming a product mimic into a Michaelis-like complex, which confirms an essential role of the MA metal ion in catalysis.
Bruton tyrosine kinase (BTK) is a non-receptor tyrosine kinase crucial for relaying signals from the B cell antigen receptor (BCR) in cancerous B lymphocytes. It has been reported that mutations in this protein cause resistance to various covalent and non-covalent drugs. Therefore, we employed a computational genomic mutation screening strategy, combined with molecular simulation, to assess the impact of clinical substitutions on the structure and binding of ARQ 531(Nemtabrutinib), the most potent BTK inhibitor. Using various machine learning algorithms, 62 clinical mutations were identified as deleterious among the 82, while 11 were classified as highly destabilizing using graph signature-based methods. We selected the top mutations that are deleterious and highly destabilizing, which include L408P, Y476D, M477R, C481R, C481Y, and L542P. Molecular docking analysis revealed no significant variations in the bonding network, while molecular simulation results revealed local changes only in the dynamic behavior. The resemblance to the wild type and mutants in certain PCs (principal components) implies that specific structural aspects or dynamics remain preserved despite the mutation, and the single energetic minimum indicates a robust and dominant structural state, reinforcing that these mutations affect the protein locally but not globally. Finally, the total binding free energy (TBE) calculations revealed that ARQ531 exhibits broadly conserved binding energetics across clinically observed BTK mutants, with select variants (notably C481 substitutions) showing moderately enhanced stabilization relative to the wild type. These findings confirm that ARQ 531 remains a significant investigational therapy specifically for overcoming drug resistance in multiple leukemias and B-cell malignancies and can serve as a starting point for designing more robust and effective BTK inhibitors.
The COVID-19 pandemic, caused by SARS-CoV-2, has emerged as a global health crisis, leading to widespread morbidity and mortality, causing significant disruption to daily life and socio-economic activities worldwide. The outbreak created an urgent need for novel therapeutics and vaccines. However, several of these pharmaceutical interventions have exhibited adverse effects with prolonged usage, emphasizing the need for alternative or complementary therapeutic approaches. Withania somnifera (known as Ashwagandha) has gained attention due to its long history of use in traditional medicine and its reported antiviral, immunomodulatory, and anti-inflammatory properties. This study investigates the binding affinities and molecular interactions of particularly three bioactive compounds in detail - Ashwagandhanolide, Sitoindoside IX and Withanolide D - using molecular docking techniques. The analysis revealed binding affinities of -9.5 kcal/mol, -8.8 kcal/mol and -8.9 kcal/mol, respectively. Detailed interaction profiles identified ADMET profiling pharmacophore modeling, van der Waals forces, hydrogen bonding, and hydrophobic interactions as key contributors to drug-likeness and stability of these compounds. These findings provide insights into the compounds' potential pharmacological applications and establish a foundation for experimental validation in the future.
Proliferating cell nuclear antigen (PCNA) protein is an emerging therapeutic target for several diseases including cancers, and certain viral infections. It was previously regarded as being undruggable, due to its functions as a molecular hub that regulates a multitude of essential cellular pathways. However, we recently developed a PCNA inhibitor, AOH1996, which acts as a molecular glue between PCNA and RNA Pol II promoting selective killing of cancer cells through the induction of transcription-replication conflicts. We have now moved this investigational new drug into Phase 1 clinical trials. Yet, the discovery of new PCNA inhibitors is still of notable importance, because of the potential for further improving the potency and overall in vivo stability relative to AOH1996, and to potentially provide more treatment options for differing patient populations and for overcoming drug resistance mechanisms that may arise from AOH1996 treatment. Moreover, targeting other PCNA mediated disease states could require the identification of novel compounds that have non-identical mechanisms of action or differing pharmokinetic and pharmodynamic profiles. We coupled artificial intelligence-based computational screens with thermal shift assays in a search for novel hit compounds, and characterized a key hit through protein crystallography, a cellular thermal shift assay and protein frustration calculations. Notably, we discovered that the Hsp90α inhibitor, SNX-2112, binds to PCNA within the major PIP-box binding pocket, revealing a new chemical scaffold for the potential development of novel PCNA-targeting inhibitors.
Water molecules play a vital role in stabilizing ribonucleic acid (RNA) structures, not only as a solvent but as active participants in molecular recognition. Yet how hydration patterns encode binding specificity across RNA conformations remains poorly understood. The human immunodeficiency virus (HIV) Rev response element (RRE) provides a model system to address this question, as it adopts multiple conformations that engage distinct peptide partners. Here, the hydration landscapes of three RNA conformations are deciphered, and the role of water in RNA recognition is studied by mapping persistent water-binding sites and quantifying hydrogen-bond lifetime using all-atom molecular dynamics simulations. The apo state exhibits a diffuse hydration shell that uniformly stabilizes the backbone. By contrast, the Rev-binding conformation exhibits a reorganized and transient hydration environment, indicating that recognition involves the adaptive restructuring of the solvent network. In yet another manner, the RSG 1.2-binding conformation retains a highly ordered hydration scaffold that persists even without the peptide, consistent with a pre-organized recognition mechanism. Together, these results reveal that hydration encodes RNA adaptability, recognition readiness, and dictates whether recognition proceeds by induced-fit or pre-organized pathways. Importantly, these findings align with previous thermodynamic analyses reporting that RRE-Rev binding is driven by structural rearrangement and solvent displacement rather than direct enthalpic contacts, underscoring the central role of water in RNA adaptability and molecular recognition.
Arsenic trioxide (As2O3) has been useful to treat acute promyelocytic leukaemia, as it inhibits the growth of various tumour cell lines and promotes apoptosis. However, its employment has been hindered by its cardiotoxic potential, since it prolongs cardiac repolarization. The transient outward K+ current (Ito) in the heart is a critical component of the early repolarization phase and a plateau of the cardiac action potential. While the molecular changes that explain the chronic effects of As2O3 on cardiac K+ currents are known, its immediate repercussions remain elusive. In the present work, we characterize the acute electrophysiological effects of As2O3 on the Ito in rat myocytes and on Kv4.3 channels co-expressed with their auxiliary subunit KChIP2c, which constitute the principal mediator of the Ito in the human ventricle. We found that this anion causes a reversible, voltage and concentration dependent inhibition (IC50 ≈ 4 μM), reaching a maximum (38% block) at 100 μM, on both cell types. Bath perfusion of 4 μM As2O3 had no significant effect on the current kinetics or on the steady-state inactivation of the Ito, but it shifted the steady-state tail activation curve to more depolarized potentials. The development of As2O3 inhibition was fourfold faster upon intracellular administration. A ligand and structure-based computational strategy including pharmacophore and molecular docking was implemented to identify the stereochemistry of the ligand-protein binding.
BCL6 plays significant roles in various cellular processes and malignancies such as diffuse large B-cell lymphoma. BCL6 performs its functions through binding of its BTB domain to different corepressors. Thus, analyzing the possible structural consequences of nsSNPs on the function of this domain would be imperative. To this end, we have selected the most deleterious SNPs of BCL6 based on various scoring algorithms. Then the selected mutations were modeled, analyzed for various physicochemical and stability properties, and used for molecular docking with the BCoR, NCoR, and SMRT. The obtained complexes were used for the calculation of binding energy and depiction of 2D interaction plots. The docked complexes were also subjected to Molecular Dynamics (MD) simulations to screen their behavior in physiological conditions. The BCL6 SNPs were filtered to 54 nsSNPs of the BTB domain. Using various tools, these nsSNPs were narrowed down to the Q113K, V105G, I78T, and I60T mutations based on their deleteriousness and stability scores. Docking analyses indicated that the exerted mutations mostly reduced the binding affinity, and the MD simulations showed the lower stability of the mutated BCL6 forms during the simulation. Given the attained results, it could be concluded that selected nsSNPs could lead to impaired BCL6 transcriptional repressive function due to loss of stability and binding affinity towards its corepressors. These observations can explain various biological or clinical differences in individuals carrying these SNPs and help with the rational design of novel personalized therapeutics. The results found by computer simulations are suggesting new experiments that need to be done in the future to prove that they are biologically and clinically applicable.
Induced-proximity therapeutics have emerged as a transformative paradigm in chemical biology and drug discovery, enabling selective control of cellular processes beyond conventional inhibitors. Between 2020 and 2025, major progress has been achieved across five modalities: proteolysis-targeting chimeras (PROTACs), molecular glues, lysosome-targeting chimeras (LYTACs), autophagy-targeting chimeras (AUTACs) and related tethering strategies, and ribonuclease-targeting chimeras (RIBOTACs). Each exploits endogenous degradation or regulatory pathways using chemically engineered bifunctional or monofunctional small molecules, thereby expanding the druggable proteome and transcriptome. This review provides a comparative analysis of their underlying organic chemistry, design principles, and mechanistic diversity. We highlight structure activity relationships, linker optimization, and chemical motifs that govern induced proximity and degradation efficiency. Advances in ligand discovery, modular synthetic methodologies, and strategies to improve pharmacokinetics and tissue selectivity are emphasized. Schematic diagrams illustrate key mechanistic steps, offering a visual framework for comparing similarities and differences across approaches. While prior reviews have focused on mechanistic and pharmacological aspects, our perspective emphasizes synthetic strategies, linker chemistry, SAR studies, and ligand optimization principles that underpin each degrader class. We examine how advances in synthetic design, modular assembly, and chemical reprogramming of ligases or receptors have broadened therapeutic potential. By critically assessing strengths, limitations, and chemical challenges across modalities, we propose a unifying organic chemistry perspective that distinguishes induced-proximity strategies from conventional small-molecule inhibition and outlines future opportunities in degrader design.
Tumor Necrosis Factor alpha (TNFα) is a pro-inflammatory cytokine critical for regulating cell survival and death. Under pathological conditions, excessive TNFα activity can lead to chronic inflammation, contributing to diseases such as inflammatory bowel disease and other autoimmune disorders. While structural studies have elucidated the atomistic details of TNFα binding to its receptor, TNF Receptor 1 (TNFR1), the influence of the membrane environment on this interaction remains poorly characterized experimentally. In this study, we employed advanced all-atom Gaussian accelerated molecular dynamics simulations to investigate how lipid-mediated interactions modulate the TNFα-TNFR1 complex. We identified key residues on both the cytokine and its receptor that govern trimer assembly, receptor binding, and potential pathological alterations. Our analysis confirmed previously identified functional sites and revealed new residues likely to contribute to the structural stability and dynamics of the complex. These findings provide a more comprehensive understanding of the molecular determinants of TNF signaling and offer a foundation for future experimental investigations into the receptor-ligand interface and membrane-mediated regulation.
The 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) cycle in thaumarchaeota contributes significantly to global organic carbon fixation as the most energetically efficient aerobic carbon fixation pathway. The thaumarchaeal 3-Hydroxypropionyl-CoA Synthetase (ADP-forming; Nmar_1309) is crucial to this efficiency, utilizing ATP to ADP catalysis. This first reported structure of Nmar_1309 reveals a homodimer with a unique domain organization and a distinct linker between subdomains 4 and 1. This structure includes the bound substrates 3HP, non-hydrolyzable ATP (ADPNP), and a phosphate which suggest an intermediate state mimicking the non-covalent interaction between 3-hydroxypropionyl-phosphate and the active site histidine prior to reaction with Coenzyme-A. Conformational differences were observed between the two chains of the homodimer, likely influenced by the binding of a single ADPNP molecule in one chain. Phylogenetic analysis suggests that while 4HB synthetases may have evolved earlier in the evolutionary timeline, 3HP synthetases in Thaumarchaeota may have occurred after the Great Oxygenation Event. These structural data provide further characterization of the 3HP/4HB cycle and, in conjunction with the structure of 4-hydroxybutyryl-CoA synthetase, Nmar_0206, provide baseline structures of the key ADP-forming Acyl-CoA synthetases within this pathway.
Escalating antimicrobial resistance necessitates the development of alternative therapeutics that circumvent conventional enzymatic and efflux-based defence systems. Antimicrobial peptides (AMPs) represent a compelling class of innate immune effectors, however, their clinical translation is hindered by incomplete mechanistic understanding of how structural organization and conformational dynamics shape antimicrobial function. In this study, we performed an integrated comparative analysis of three mechanistically representative AMPs-LL-37, HNP-1, and magainin-2-to resolve how maturation pathways, fold topology, amphipathic architecture, and dynamic target engagement govern antimicrobial action. Consensus secondary-structure prediction, AlphaFold2/PEP-FOLD modelling, and physicochemical profiling revealed three distinct structural signatures. LL-37 exhibited a flexible disorder-to-helix transition enabling adaptive, curvature-driven membrane dissolution, HNP-1 adopted a rigid cysteine-stabilized β-sheet that promotes lipid clustering and entropic inhibition of membrane-associated enzymes, and magainin-2 formed a stable amphipathic α-helix optimized for toroidal pore initiation. Machine-learning classification corroborated strong antimicrobial likelihood for HNP-1 and magainin-2, with LL-37 displaying context-dependent activation. Protein-peptide docking and normal-mode elastic network modelling further demonstrated the possibility of LL-37 allosterically dampening conformational cycling of the MexB efflux pump, HNP-1 restricting catalytic-loop mobility in LpxC, and magainin-2 enhancing correlated β-barrel breathing in OprF to promote pore formation. These findings delineate three mechanistically distinct antimicrobial strategies-adaptive membrane dissolution, rigid pore-stacking inhibition, and dynamic pore initiation-linked directly to peptide structural organization. This framework provides a rational basis for mechanism-guided AMP optimization and the engineering of next-generation membrane-active therapeutics with reduced resistance susceptibility.
Bacterial cell poles play a fundamental role in several cellular processes, such as cell cycle, chemotaxis, cell differentiation, development, growth, and structure of the bacterial cell, as well as protein localization. Using a set of bioinformatics tools, we evaluated the probability of 19 bacterial cell pole-related proteins to be disordered and undergo spontaneous liquid-liquid phase separation (LLPS). Our analysis revealed that11 cell pole-related proteins are predicted to be highly disordered, 7 proteins are moderately disordered, and only one protein is expected to be highly ordered. Furthermore, this intrinsic disorder propensity is mostly evolutionary conserved. Most of the analyzed 19 cell pole-related proteins were found to be associated with the LLPS process, with TipN, PopZ, and PBP2A being capable of spontaneous phase separation, RacA, Noc, PBP1A/1B, ParB, PBP3, PBP2B, FtsZ, MipZ, MinD, and MreB being expected to potentially serve as droplet clients, and with the remaining proteins (DivIVA, ComN, Maf, PBP4, MinC, and MinJ) being predicted to be unrelated to LLPS. The results suggested that FtsZ, DivIVA, and MiPZ serve as the main regulatory proteins, being well-known for their role in forming the septum and chromosomal segregation. Furthermore, PopZ and TipN proteins contribute to high stress resistance. Clarifying the function and effects of each mechanism gives insight into the organization of bacterial cells and some strategies for antimicrobial targets.
The tyrosinase enzyme plays a pivotal role in melanin pigment production; however, heightened tyrosinase activity can lead to undesired pigmentation. Consequently, inhibiting this enzyme's function stands as a critical research avenue for devising effective strategies to mitigate pigmentation issues. This study aimed to forecast the biological activity of chemical compounds capable of inhibiting tyrosinase and elucidate pivotal elements influencing this enzyme's activity. To achieve this goal, we employed computational techniques to construct a model predicting the biological activity of these compounds. Initially, we identified 27 tyrosinase inhibitors from previous studies. Subsequently, after performing ADMET studies, we extracted and pre-processed the significant features of each compound to develop a Stepwise-MLR model. Moreover, with the help of this model, we were able to identify the most influential and novel structural features that directly affect enzyme activity and determine the importance factor of each feature. Furthermore, all derived inhibitors with evaluated inhibition constants were docked to the active site of target tyrosinase to investigate the binding mode of the compounds. Docking analysis indicated T1 as the most stable compound with a binding energy of -8.00 kcal/mol. T1 as the most active compound identified through these computational studies can be applied as a prospective tyrosinase inhibitor. The implications of our findings extend to the development of new therapies for pigmentation disorders, notably within the cosmetic and dermatological sectors.
The CRISPR/Cas system is a potential tool for genome editing, yet it faces challenges due to off-target activity caused by mismatches at specific positions. However, Off-target activity can be minimized by optimal design of guide RNA (gRNA) but there remains a possibility of unintended cleavage, highlighting the role of the Cas nuclease in off-target recognition and binding the target site. This study focuses on comparing the conformational dynamics and stability of Wildtype, RR, RVR, RRm and RVRm variants of AsCas12a with gRNA-DNA bound complexes. It was found that the cross-correlation coefficient between His1167 of the NUC domain and Thr384 of the REC II domain significantly increased after the K949A mutation compared to other variants. The extensive spread of principal components also revealed flexibility in both Cas nuclease and gRNA-DNA hybrid of RVR variant and wildtype AsCas12a whereas the confined clusters in PCA plot suggests increased stability in both the variants after mutation. This study shows the role of K949A mutation in improving stability of PAM variants and predicted critical residues such as His1167, Thr384 and Ser959, in inducing stability in mutants of PAM variants.
