Analysis Of Binding Interactions Research Articles

Computational Analysis of Lenalidomide and Pomalidomide Enantiomers' Binding Interactions With Prostaglandin (PG)-Protein: Implications for Inflammatory Activity in Cancer.

Lenalidomide and Pomalidomide are chiral immunomodulatory drugs (IMiDs) and have antiangiogenic and anti-immunomodulatory activity. Each enantiomer may have distinct binding and biological activity. This study aimed to explore the in-silico binding of both enantiomers of Lenalidomide and Pomalidomide with Prostaglandin and its potential impact on persisting inflammatory activity in cancer. This can further provide insight into the transport of pro-inflammatory mediators and their potential implications for the inflammatory microenvironment within tumors. Molecular docking studies were performed to explore the binding potential of both enantiomers of Lenalidomide and Pomalidomide with Pg protein. The crystal structure of Pg-protein (PDB ID: 1IW7) was obtained from the Protein Data Bank. The binding energies for (-)-Lenalidomide and (+)-Lenalidomide were -6.7 and -7.2 kcal/mol, respectively, while the binding energies for (-)-Pomalidomide and (+)-Pomalidomide were -7.8 and -8.1 kcal/mol, respectively. The binding mode analysis revealed that all four compounds formed hydrogen bonds with key amino acid residues of Pg-protein. The hydrogen bond distances for (-)-Lenalidomide, (+)-Lenalidomide, (-)-Pomalidomide, and (+)-Pomalidomide were 2.1 Å, 2.0 Å, 2.2 Å, and 2.1 Å, respectively. The present study suggests that both enantiomers of Lenalidomide and Pomalidomide have a high affinity for Pg-protein and can effectively target the Pg-protein pathway to persist inflammatory activity in cancer.By targeting inflammation-mediated processes, these drugs may offer a novel strategy to combat tumor progression.

Computational exploration of FOXM1 inhibitors for glioblastoma: an integrated virtual screening and molecular dynamics simulation study

In this study, a comprehensive investigation of a set of phytochemicals to identify potential inhibitors for the Forkhead box protein M1 (FOXM1) was conducted. FOXM1 is overexpressed in glioblastoma (GBM) cells and plays a crucial role in cell cycle progression, proliferation, and invasion. FOXM1 inhibitors have shown promising results in preclinical studies, and ongoing clinical trials are assessing their efficacy in GBM patients. However, there are limited studies on the identification of novel compounds against this attractive therapeutic target. To address this, the NPACT database containing 1,574 phytochemicals was used, employing a hierarchical multistep docking approach, followed by an estimation of relative binding free energy. By fixing user-defined XP-dock and MM-GBSA cut-off scores of −6.096 and −37.881 kcal/mol, the chemical space was further narrowed. Through exhaustive analysis of molecular binding interactions and various pharmacokinetics profiles, we identified four compounds, namely NPACT00002, NPACT01454, NPACT00856, and NPACT01417, as potential FOXM1 inhibitors. To assess the stability of protein-ligand binding in dynamic conditions, 100 ns Molecular dynamics (MD) simulations studies were performed. Furthermore, Molecular mechanics with generalized Born and surface area solvation (MM-GBSA) based binding free energy estimations of the entire simulation trajectories revealed a strong binding affinity of all identified compounds towards FOXM1, surpassing that of the control drug Troglitazone. Based on extensively studied multistep docking approaches, we propose that these molecules hold promise as FOXM1 inhibitors for potential therapeutic applications in GBM. However, experimental validation will be necessary to confirm their efficacy as targeted therapies. Communicated by Ramaswamy H. Sarma

Molecular Docking Studies of selected Erythrina variegata Leaves Alkaloids towards Estrogen Receptor (1A52 and 1GWR) and their Binding Interaction Analysis

Medicinal plants possess many phytochemicals of great therapeutic value and many of them are effective in killing cancer cells. These compounds working by variety of mechanisms and in most of the cases they exhibit their anticancer potentiality by inhibiting many proteins involved in cell growth and division. Molecular docking is a computational approach which facilitates the finding of the best molecule from a group which may bind with the highest affinity with the intended target by providing a virtual biological system. This process works on the basis of specific algorithm and involves scoring function to rank the molecules that fit with the target. The present study has been designed to investigate the potentiality of eight alkaloids compounds from leaves of Erythrina variegata natural products and two anticancer drugs have selected and docked against Estrogen receptor proteins (PDB ID:1A52 and 1GWR) to treat cancer cells. Among them 6-hydroxy genistein ranks first with very good binding with the very good dock score with these receptors and has the potential to treat the cancer cells against the Estrogen receptor proteins 1A52 and 1GWR.

The molecular mechanism of the effects of the anti-neuropathic ligands on the modulation of the Sigma-2 receptor: An in-silico study

Neuropathic pain (NP) is a prevalent medical condition that lacks an effective treatment. Recently, the Sigma-2 receptor (S2R) has been proposed as a potential therapeutic target for NP. Some highly-selective S2R ligands (UKH1114, CM398, and YTD) have shown promising results in vivo, but the molecular interaction between the S2R and these ligands has been scarcely investigated. This work explores changes in the S2R upon interaction with the three mentioned ligands using in silico approaches. The results indicated that the ICL1, H1, ICL2, and ECL are the most dynamic regions of S2R in all systems. Binding interaction analysis identified amino acids with significant contribution to the binding free energy. Notably, the UKH1114-S2R simulation trajectory revealed that small alterations in the ICL1, H1, ICL2, and ECL form a new stable opening in the S2R, linking the occluded S2R binding pocket to the endoplasmic reticulum lumen, providing more evidence for the assumptions about the EBP and S2R mechanism of function. Further, the agreement between the membrane parameters in our study and experimental values confirms the validity of the MD simulations. Overall, this study provides new insights into the interaction between anti-NP ligands and the S2R.

Framing a novel holistic energy subsystem structure for water-energy-food nexus based on existing literature (basic concepts)

It is interesting to note that the country of Iran is essential in terms of energy production and consumption, and the economy of Iran is mainly dependent on energy revenues. Therefore, thermal and hydropower plants consume water to produce various energy carriers. Considering that Iran is suffering from water stress, the nexus of water and energy becomes very important. This paper frames a comprehensive structure for Iran's energy subsystem within the Water, Energy, and Food (WEF) nexus system. The energy subsystem's supply and demand side in the proposed framework are formulated using data and physic-based equations. The presented framework addresses most interactions between WEF subsystems in a dynamic and adaptive setting. It is shown that through analysis of binding interactions between WEF, different management scenarios can boost the flexibility of the supply and demand side of the energy subsystem. In addition, by incorporating this framework, the water subsystem will manage the allocated and consumed water on the supply side and arrive at the most desirable outcome for the water sector. Also, the optimal cropping pattern could be evaluated based on energy consumption.

Activation of Human CD8+ T Cells with Nitroso Dapsone-Modified HLA-B*13:01-Binding Peptides.

Previous studies have shown that cysteine-reactive drug metabolites bind covalently with protein to activate patient T cells. However, the nature of the antigenic determinants that interact with HLA and whether T cell stimulatory peptides contain the bound drug metabolite has not been defined. Because susceptibility to dapsone hypersensitivity is associated with the expression of HLA-B*13:01, we have designed and synthesized nitroso dapsone-modified, HLA-B*13:01 binding peptides and explored their immunogenicity using T cells from hypersensitive human patients. Cysteine-containing 9-mer peptides with high binding affinity to HLA-B*13:01 were designed (AQDCEAAAL [Pep1], AQDACEAAL [Pep2], and AQDAEACAL [Pep3]), and the cysteine residue was modified with nitroso dapsone. CD8+ T cell clones were generated and characterized in terms of phenotype, function, and cross-reactivity. Autologous APCs and C1R cells expressing HLA-B*13:01 were used to determine HLA restriction. Mass spectrometry confirmed that nitroso dapsone-peptides were modified at the appropriate site and were free of soluble dapsone and nitroso dapsone. APC HLA-B*13:01-restricted nitroso dapsone-modified Pep1- (n = 124) and Pep3-responsive (n = 48) CD8+ clones were generated. Clones proliferated and secreted effector molecules with graded concentrations of nitroso dapsone-modified Pep1 or Pep3. They also displayed reactivity against soluble nitroso dapsone, which forms adducts in situ, but not with the unmodified peptide or dapsone. Cross-reactivity was observed between nitroso dapsone-modified peptides with cysteine residues in different positions in the peptide sequence. These data characterize a drug metabolite hapten CD8+ T cell response in an HLA risk allele-restricted form of drug hypersensitivity and provide a framework for structural analysis of hapten HLA binding interactions.

Phenolic compounds as histone deacetylase inhibitors: binding propensity and interaction insights from molecular docking and dynamics simulations.

Histone deacetylases are well-established target enzymes involved in the pathology of different diseases including cancer and neurodegenerative disorders. The approved HDAC inhibitor drugs are associated with cellular toxicities. Different phenolic compounds have been shown to possess inhibitory activities against HDACs and are, therefore, considered safer alternatives to synthetic compounds. Here, we elucidated the binding mode and calculated the binding propensity of some of the top phenolic compounds against different isoforms representing different classes of Zn2+ ion-containing HDACs using the molecular docking approach. Our data reaffirmed the activity of the studied phenolic compounds against HDACs. Binding interaction analysis suggested that these compounds can block the activity of HDACs with or without binding to the active site zinc metal ion. Furthermore, molecular dynamics (MD) simulations were carried out on the selected crystal and docking complexes of each selected HDAC isoform. Analysis of root-mean-square displacement (RMSD) showed that the phenolic compounds demonstrated a stable binding mode over 50ns in a way that is comparable to the cocrystal ligands. Together, these findings can aid future efforts in the search for natural inhibitors of HDACs.

Investigations on a mononuclear Cu(ii) Schiff base complex: theoretical calculations, catechol oxidase activity, and protein binding interaction analysis

A new mononuclear Cu(ii) complex has been assessed in detail using experimental and theoretical calculations and explored as a model for catecholase activity and protein binding (BSA & HSA) interaction analysis.

Diversifying the chloroquinoline scaffold against SARS-COV-2 main protease: Virtual screening approach using cross-docking, sitemap analysis and molecular dynamics simulation

The absence of designated remedies for coronavirus disease 19 (Covid-19) and the lack of treatment protocols drove scientists to propose new small molecules and to attempt to repurpose existing drugs against various targets of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in order to bring forward efficient solutions. The main protease (Mpro) is one of the most promising drug targets due to its crucial role in fighting viral replication. Several antiviral drugs have been used in an attempt to overcome the pandemic, such as hydroxychloroquine (HCQ). Despite its perceived positive outcomes in the beginning of the disease, HCQ was associated with several drawbacks, such as insolubility, toxicity, and cardiac adverse effects. Therefore, in the present study, a structure-based virtual screening approach was performed to identify structurally modified ligands of the chloroquinoline (CQ) scaffold with good solubility, absorption, and permeation aimed at eventually suggesting a more dependable alternative. PDB ID:7BRP Mpro was chosen as the most reliable receptor after cross-docking calculation using 30 crystal structures. Then, a SiteMap analysis was performed and a total of 231,456 structurally modified compounds of the CQ scaffold were suggested. After Lipinski criteria filtration, 64,312 molecules were docked and their MM-GBSA free binding energy were calculated. Next, ADME descriptors were calculated, and 12 molecules with ADME properties better than that of HCQ were identified. The resulting molecules were subjected to molecular dynamics (MD) simulation for 100 ns. The results of the study indicate that 3 molecules (CQ_22; CQ_2 and CQ_5) show better interactions and stability with the Mpro receptor. Binding interaction analysis indicates that GLU143, THR26, and HIS41 amino acids are potential binding hot-spot residues for the remaining 3 ligands.

Identification of Novel Ribonucleotide Reductase Inhibitors for Therapeutic Application in Bile Tract Cancer: An Advanced Pharmacoinformatics Study

Biliary tract cancer (BTC) is constituted by a heterogeneous group of malignant tumors that may develop in the biliary tract, and it is the second most common liver cancer. Human ribonucleotide reductase M1 (hRRM1) has already been proven to be a potential BTC target. In the current study, a de novo design approach was used to generate novel and effective chemical therapeutics for BTC. A set of comprehensive pharmacoinformatics approaches was implemented and, finally, seventeen potential molecules were found to be effective for the modulation of hRRM1 activity. Molecular docking, negative image-based ShaEP scoring, absolute binding free energy, in silico pharmacokinetics, and toxicity assessments corroborated the potentiality of the selected molecules. Almost all molecules showed higher affinity in comparison to gemcitabine and naphthyl salicylic acyl hydrazone (NSAH). On binding interaction analysis, a number of critical amino acids was found to hold the molecules at the active site cavity. The molecular dynamics (MD) simulation study also indicated the stability between protein and ligands. High negative MM-GBSA (molecular mechanics generalized Born and surface area) binding free energy indicated the potentiality of the molecules. Therefore, the proposed molecules might have the potential to be effective therapeutics for the management of BTC.

Sesquiterpene lactones from Artemisia vulgaris L. as potential NO inhibitors in LPS-induced RAW264.7 macrophage cells.

Twelve new guaianolide sesquiterpene lactones (1–12), along with ten known analogs (13–22) were isolated from an EtOH extract of the dried aerial parts of Artemisia vulgaris L. The new structures were elucidated via abundant spectroscopic data analyses (HRESIMS, IR, 1D, and 2D NMR), and the absolute configurations of these compounds were determined by X-ray crystallography and ECD calculations. The compounds (1−22) were identified as guaiane-type sesquiterpenes with characteristic α-methylene-γ-lactone and α,β-unsaturated carbonyl moieties. All compounds were tested for their inhibitory activity against NO production in lipopolysaccharide-stimulated RAW264.7 macrophages. The isolated sesquiterpenoids dose-dependently exhibited an NO production inhibitory activity by inhibiting the expression of inducible NO oxidase (iNOS) and cyclooxygenase-2 (COX-2) with IC50 values ranging from 1.0 to 3.6 μM. The inhibitory effect on the NO production of the compounds (1–4 and 6–22) is better than that of the positive control (dexamethasone). The different substitutions of compounds on C-8 influence anti-inflammatory effects, as evidenced by the in silico analysis of related binding interactions of new compounds (1–12) with iNOS.

Model for Calcium‐Dependent Activation of a Type I Metacaspase From the Fungus Schizophyllum commune

Apoptosis or programmed cell death (PCD) is one of the most important cellular processes which functions to avoid the accumulation of mutations or damage within a cell. PCD has been extensively studied in eukaryotic cells, demonstrating that caspase proteins have a significant role in this process. However, unlike mammalian cells, caspase genes are absent in plants, fungi, and protozoa. Instead, these organisms contain homologous proteins known as metacaspases which have demonstrated similar proteolytic functions in aiding PCD. Determining an accurate model for how these metacaspases are activated could provide insight into the methods behind this protease‐dependent cell death which could lead to further strategies for the regulation of fungal infections. This research focuses on the model of activation for the Type I metacaspases from the fungus Schizophyllum commune. The S. commune Type I metacaspases are dependent on calcium ions for activation; however, the molecular mechanism behind how calcium activates this protease remains unknown.It has been proposed that when calcium binds to the metacapsape, these ions induce a structural rearrangement of the active‐site region leading to activation. To further understand how calcium and other divalent metallic cations influence the activation of this protease, we used kinetic activity assays to quantify metacaspase activity and differential scanning fluorimetry to measure divalent metallic cation‐metacaspase binding.In addition to strong activation by calcium, manganese ions have demonstrated a moderate effect on metaspace activity, while magnesium, copper, samarium, and cobalt don’t affect activity. We will present evidence that calcium binds to two sites on the protein, while manganese binds to only a single site. Furthermore, calcium binding to the Type I metacapses occurs at two sites with affinities that differ by several orders of magnitude. Initial results suggest that it is necessary for calcium to saturate the high affinity site prior to binding to the low affinity site. Thus, it has been predicted that calcium’s initial saturation of the high affinity site induces slight conformation changes to the active‐site; however, once the low affinity site has been saturated, an active‐site loop undergoes further rearrangements resulting in metacaspase activation. Further analysis of binding interactions between the Type I metacapse and calcium (along with other divalent cations) will provide insight into how these structural rearrangements lead to increased metacaspase activity.

Exploring the structure function relationship of heme peroxidases: Molecular dynamics study on cytochrome c peroxidase variants

Cytochrome c peroxidase (Ccp1) is a mitochondrial heme-containing enzyme that has served for decades as a chemical model to explore the structure function relationship of heme enzymes. Unveiling the impact of its heme pocket residues on the structural behavior, the non-covalent interactions and consequently its peroxidase activity has been a matter of increasing interest. To further probe these roles, we conducted intensive all-atom molecular dynamics simulations on WT and nineteen in-silico generated Ccp1 variants followed by a detailed structural and energetic analysis of H2O2 binding and pairwise interactions. Different structural analysis including RMSD, RMSF, radius of gyration and the number of Hydrogen bonds clearly demonstrate that none of the studied mutants induce a significant structural change relative to the WT behavior. In an excellent agreement with experimental observations, the structural change induced by all the studied mutant systems is found to be very localized only to their surrounding environment. The determined interaction energies between residues and Gibbs binding energies for the WT Ccp1 and the nineteen variants, helped to identify the precise effect of each mutated residues on both the binding of H2O2 and the non-covalent interaction and thus the overall peroxidase activity. The roles of surrounding residues in adopting unique distinctive electronic feature by Ccp1 has been discerned. Our valuable findings have clarified the functions of various residues in Ccp1 and thereby provided novel atomistic insights into its function. Overall, due to the conserved residues of the heme-pocket amongst various peroxidases, the obtained remarks in this work are highly valuable.

Binding Interaction Analysis of Phytoconstituents of Commiphora mukul with 3CLPro and PlPro Enzymes of SARS-CoV-2 Virus

The SARS-CoV-2 belongs to a family of coronaviruses, responsible for the COVID-19 pandemic. The Chymotrypsin-like protease (3CLpro) and Papain-like protease (PLPro), two important enzymes of the SARS-CoV-2, play a key role in translating the viral RNA genome into functional proteins. This study aimed to evaluate and analyze the binding interactions of phytochemicals of oleoresin of Commiphora mukul plant with 3CLPro and PLPro enzymes of SARS-CoV-2 virus. Docking studies were performed on 3CLPro and PLPro with 30 phytoconstituents using AutoDock Vina. A total of 12 compounds were shortlisted based on their minimum binding energies. Their minimum binding energy ranged from -9.2 to -7.1 kcal and the analysis and interpretations are tabulated. Further, the ADME, drug-likeness, and bioactivity scores were documented. The current study enlightened the binding abilities, and the interactions of constituents present in the oleoresin of Commiphora mukul plant with the active sites of 3CLPro and PLPro enzymes.

Genome-wide Profiling of Transcription Factor-DNA Binding Interactions in Candida albicans: A Comprehensive CUT&RUN Method and Data Analysis Workflow.

Regulatory transcription factors control many important biological processes, including cellular differentiation, responses to environmental perturbations and stresses, and host-pathogen interactions. Determining the genome-wide binding of regulatory transcription factors to DNA is essential to understanding the function of transcription factors in these often complex biological processes. Cleavage under targets and release using nuclease (CUT&RUN) is a modern method for genome-wide mapping of in vivo protein-DNA binding interactions that is an attractive alternative to the traditional and widely used chromatin immunoprecipitation followed by sequencing (ChIP-seq) method. CUT&RUN is amenable to a higher-throughput experimental setup and has a substantially higher dynamic range with lower per-sample sequencing costs than ChIP-seq. Here, a comprehensive CUT&RUN protocol and accompanying data analysis workflow tailored for genome-wide analysis of transcription factor-DNA binding interactions in the human fungal pathogen Candida albicans are described. This detailed protocol includes all necessary experimental procedures, from epitope tagging of transcription factor-coding genes to library preparation for sequencing; additionally, it includes a customized computational workflow for CUT&RUN data analysis.

Evaluation of the binding performance of flavonoids to estrogen receptor alpha by Autodock, Autodock Vina and Surflex-Dock

Molecular docking is a widely used method to predict the binding modes of small-molecule ligands to the target binding site. However, it remains a challenge to identify the correct binding conformation and the corresponding binding affinity for a series of structurally similar ligands, especially those with weak binding. An understanding of the various relative attributes of popular docking programs is required to ensure a successful docking outcome. In this study, we systematically compared the performance of three popular docking programs, Autodock, Autodock Vina, and Surflex-Dock for a series of structurally similar weekly binding flavonoids (22) binding to the estrogen receptor alpha (ERα). For these flavonoids-ERα interactions, Surflex-Dock showed higher accuracy than Autodock and Autodock Vina. The hydrogen bond overweighting by Autodock and Autodock Vina led to incorrect binding results, while Surflex-Dock effectively balanced both hydrogen bond and hydrophobic interactions. Moreover, the selection of initial receptor structure is critical as it influences the docking conformations of flavonoids-ERα complexes. The flexible docking method failed to further improve the docking accuracy of the semi-flexible docking method for such chemicals. In addition, binding interaction analysis revealed that 8 residues, including Ala350, Glu353, Leu387, Arg394, Phe404, Gly521, His524, and Leu525, are the key residues in ERα-flavonoids complexes. This work provides reference for assessing molecular interactions between ERα and flavonoid-like chemicals and provides instructive information for other environmental chemicals.

Synthesis, Molecular Docking and DFT Studies of Biologically Active N-((3-(4-Nitrophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)aniline Derivatives

Some biologically active pyrazole clubbed imino molecules have been designed and synthesized from 1-phenyl-3-nitro phenyl-1H- pyrazol-4-carboxaldehyde and substituted aromatic amines via acid catalyzed condensation reaction. All the synthesized molecules were characterized by IR, 1H NMR, 13C NMR and mass spectral techniques. The in vitro antibactericidal property of the synthesized compounds was screened and compared with the results of theoretical molecular docking. Optimization of molecular geometry, DNA binding interaction and FMO analysis were also investigated by computational studies using Gaussian 16 package at B3LYP/6-31G(d,p) level. All the synthesized compounds exhibited moderate to good biological activities both experimentally and theoretically.

A Comprehensive Review on the Interaction of Milk Protein Concentrates with Plant-Based Polyphenolics.

Functional properties and biological activities of plant-derived polyphenolic compounds have gained great interest due to their epidemiologically proven health benefits and diverse industrial applications in the food and pharmaceutical industry. Moreover, the food processing conditions and certain chemical reactions such as pigmentation, acylation, hydroxylation, and glycosylation can also cause alteration in the stability, antioxidant activity, and structural characteristics of the polyphenolic compounds. Since the (poly)phenols are highly reactive, to overcome these problems, the formulation of a complex of polyphenolic compounds with natural biopolymers is an effective approach. Besides, to increase the bioavailability and bioaccessibility of polyphenolic compounds, milk proteins such as whey protein concentrate, sodium caseinate, and milk protein concentrate act as natural vehicles, due to their specific structural and functional properties with high nutritional value. Therefore, milk proteins are suitable for the delivery of polyphenols to parts of the gastrointestinal tract. Therefore, this review reports on types of (poly)phenols, methods for the analysis of binding interactions between (poly)phenols–milk proteins, and structural changes that occur during the interaction.

Critical issues encountered in the analysis of protein-phenolic binding interactions via fluorescence spectroscopy

There is a substantial volume of research articles in the field of protein-ligand interactions, with a large proportion using inappropriate methodology for the fluorescence spectroscopy analysis of binding interactions. The most common issues being overlooked are the inner filter effect and the use of unsuitable equations to calculate binding strength and stoichiometry, leading to the propagation of questionable methodology and reported results throughout multiple fields. In this communication, we carefully explain these issues and approaches to overcome them, including accounting for the inner filter effect, the use of appropriate equations to obtain dissociation constant (Kd) values (as opposed to the commonly misused Stern-Volmer equation), and carefully dealing with binding stoichiometry using the Job plot (often misjudged using the Hill coefficient instead). We hope that the work will bring attention to critical and often common issues in fluorescence spectroscopy, as well as improving the approaches used in the binding analysis of protein-ligand systems. Thus, it should provide a good example of how to go about this type of research, as well as being of interest to the broad readership of the journal.

Computational Analysis of Binding Interactions between the Ryanodine Receptor Type 2 and Calmodulin.

Mutations in the cardiac ryanodine receptor type 2 (RyR2) have been linked to a variety of cardiac arrhythmias, such as catecholaminergic polymorphic ventricular tachycardia (CPVT). RyR2 is regulated by calmodulin (CaM), and mutations that disrupt their interaction can cause aberrant calcium release, leading to an arrhythmia. It was recently shown that increasing the RyR2-CaM binding affinity could rescue a defective CPVT-related RyR2 channel to near wild-type behavior. However, the interactions that determine the binding affinity at the RyR2-CaM binding interface are not well understood. In this study, we identify the key domains and interactions, including several new interactions, involved in the binding of CaM to RyR2. Also, our comparison between the wild-type and V3599K mutant suggests how the RyR2-CaM binding affinity can be increased via a change in the central and N-terminal lobe binding contacts for CaM. This computational approach provides new insights into the effect of a mutation at the RyR2-CaM binding interface, and it may find utility in drug design for the future treatment of cardiac arrhythmias.