D. Thirumal Kumar

Influence of V54M mutation in giant muscle protein titin: a computational screening and molecular dynamics approach

Recent genetic studies have revealed the impact of mutations in associated genes for cardiac sarcomere components leading to dilated cardiomyopathy (DCM). The cardiac sarcomere is composed of thick and thin filaments and a giant muscle protein known as titin or connectin. Titin interacts with T-cap/telethonin in the Z-line region and plays a vital role in regulating sarcomere assembly. Initially, we screened all the variants associated with giant protein titin and analyzed their impact with the aid of pathogenicity and stability prediction methods. V54M mutation found in the hydrophobic core region of the protein associated with abnormal clinical phenotype leads to DCM was selected for further analysis. To address this issue, we mapped the deleterious mutant V54M, modeled the mutant protein complex, and deciphered the impact of mutation on binding with its partner telethonin in the titin crystal structure of PDB ID: 1YA5 with the aid of docking analysis. Furthermore, two run molecular dynamics simulation was initiated to understand the mechanistic action of V54M mutation in altering the protein structure, dynamics, and stability. According to the results obtained from the repeated 50 ns trajectory files, the overall effect of V54M mutation was destabilizing and transition of bend to coil in the secondary structure was observed. Furthermore, MMPBSA elucidated that V54M found in the Z-line region of titin decreases the binding affinity of titin to Z-line proteins T-cap/telethonin thereby hindering the protein–protein interaction.

Deciphering the impact of somatic mutations in exon 20 and exon 9 of PIK3CA gene in breast tumors among Indian women through molecular dynamics approach

We examined 25 breast tumor samples for somatic mutations in exon 20 and exon 9 of PIK3CA gene in South Indian population. Genomic DNA was isolated and amplified for PIK3CA gene, followed by direct sequencing of purified polymerase chain reaction products. We identified PI3K3CA mutations in 5 of 25 (20%), including four of the mutations in p.H1047R and one in p.H1047L. Nucleotide base substitution A to G (c.3140A > G) and A to T (c.3140A > T) results in p.H1047R and p.H1047L mutation in exon 20 of PIK3CA gene. We did not observe any mutation in exon 9 of PIK3CA gene. Furthermore, we investigated the effect of mutations on protein structure and function by the combination of sequence and structure-based in silico prediction methods. This determined the underlying relationship between the mutation and its phenotypic effects. Next step, we complemented by molecular dynamics simulation analysis (30 ns) of native and mutant structures that measured the effect of mutation on protein structure. The obtained results support that the application of computational methods helps predict the biological significance of mutations.

Determining the role of missense mutations in the POU domain of HNF1A that reduce the DNA-binding affinity: A computational approach

Maturity-onset diabetes of the young type 3 (MODY3) is a non-ketotic form of diabetes associated with poor insulin secretion. Over the past years, several studies have reported the association of missense mutations in the Hepatocyte Nuclear Factor 1 Alpha (HNF1A) with MODY3. Missense mutations in the POU homeodomain (POUH) of HNF1A hinder binding to the DNA, thereby leading to a dysfunctional protein. Missense mutations of the HNF1A were retrieved from public databases and subjected to a three-step computational mutational analysis to identify the underlying mechanism. First, the pathogenicity and stability of the mutations were analyzed to determine whether they alter protein structure and function. Second, the sequence conservation and DNA-binding sites of the mutant positions were assessed; as HNF1A protein is a transcription factor. Finally, the biochemical properties of the biological system were validated using molecular dynamic simulations in Gromacs 4.6.3 package. Two arginine residues (131 and 203) in the HNF1A protein are highly conserved residues and contribute to the function of the protein. Furthermore, the R131W, R131Q, and R203C mutations were predicted to be highly deleterious by in silico tools and showed lower binding affinity with DNA when compared to the native protein using the molecular docking analysis. Triplicate runs of molecular dynamic (MD) simulations (50ns) revealed smaller changes in patterns of deviation, fluctuation, and compactness, in complexes containing the R131Q and R131W mutations, compared to complexes containing the R203C mutant complex. We observed reduction in the number of intermolecular hydrogen bonds, compactness, and electrostatic potential, as well as the loss of salt bridges, in the R203C mutant complex. Substitution of arginine with cysteine at position 203 decreases the affinity of the protein for DNA, thereby destabilizing the protein. Based on our current findings, the MD approach is an important tool for elucidating the impact and affinity of mutations in DNA-protein interactions and understanding their function.

Role of E542 and E545 missense mutations of PIK3CA in breast cancer: a comparative computational approach

Recent statistics describe breast cancer as the leading cause of death among women across the world with varied causes and reasons. Lifestyle, diet, genetic and environmental factors introduce their generous contributions towards breast cancer, among which genetic factors have lately become one of the most important aspects in understanding the mechanism. Although various genes have already been reported in causing breast cancer, PIK3CA stands second on the list. Mutations observed in this gene have the ability to trigger the different activities of the cell, thereby bypassing the regular cellular cycle. Among the mutations in PIK3CA, three hotspot mutations were commonly reported, one in the catalytic domain (position HIS1047) and other two in the helical domain (position GLU542 and GLU545). In the helical domain of PIK3CA, the lysine substitution at 542–545 positions was significantly studied in causing breast cancer. To compare the deleterious effect of these mutations, in silico prediction tools along with molecular dynamics simulations and molecular docking approach was initiated to analyse the change in binding landscape upon mutation. In this comparative analysis, we report that the mere existence of mutant E545K can trigger the function of the protein but may not be as harmful as H1047R. Among the two mutations E542K and E545K, the latter shows the most deleterious effect that correlates with the previous reported experimental studies. We assume the results observed in this combinatorial computational study might further pave a better way for providing better treatment procedures.Recent statistics describe breast cancer as the leading cause of death among women across the world with varied causes and reasons. Lifestyle, diet, genetic and environmental factors introduce their generous contributions towards breast cancer, among which genetic factors have lately become one of the most important aspects in understanding the mechanism. Although various genes have already been reported in causing breast cancer, PIK3CA stands second on the list. Mutations observed in this gene have the ability to trigger the different activities of the cell, thereby bypassing the regular cellular cycle. Among the mutations in PIK3CA, three hotspot mutations were commonly reported, one in the catalytic domain (position HIS1047) and other two in the helical domain (position GLU542 and GLU545). In the helical domain of PIK3CA, the lysine substitution at 542–545 positions was significantly studied in causing breast cancer. To compare the deleterious effect of these mutations, in silico prediction tools along...

Unraveling the inhibition mechanism of cyanidin-3-sophoroside on polyphenol oxidase and its effect on enzymatic browning of apples

The hunt for anti-browning agents in the food and agricultural industries aims to minimize nutritional loss and prolong post harvest storage. In the present study, the effect of cyanidin-3-sophoroside (CS) from Garcinia mangostana rind, on polyphenol oxidase (PPO) activity was investigated. The non-competitive inhibition mode of CS was determined by Lineweaver Burk plot. CS forms a ground-state complex by quenching the intrinsic fluorescence of PPO. The static quenching was temperature-dependent with an activation energy of 4.654±0.1091kJmol-1 to withstand the disruption of amino acid residues of the enzyme binding site. The enzyme conformational change was validated by 3D fluorescence and CD spectrum. Docking (binding energy -8.124kcal/mol) and simulation studies confirmed the binding pattern and stability. CS decreased PPO activity and browning index of fresh cut apples and prolonged the shelf life. Thus, CS appears to be a promising anti-browning agent to control enzymatic browning.

DNA Repair Gene (XRCC1) Polymorphism (Arg399Gln) Associated with Schizophrenia in South Indian Population: A Genotypic and Molecular Dynamics Study

This paper depicts the first report from an Indian population on the association between the variant Arg399Gln of XRCC1 locus in the DNA repair system and schizophrenia, the debilitating disease that affects 1% of the world population. Genotypic analysis of a total of 523 subjects (260 patients and 263 controls) revealed an overwhelming presence of Gln399Gln in the case subjects against the controls (P < 0.0068), indicating significant level of association of this nsSNP with schizophrenia; the Gln399 allele frequency was also perceptibly more in cases than in controls (p < 0.003; OR = 1.448). The results of the genotypic studies were further validated using pathogenicity and stability prediction analysis employing computational tools [I-Mutant Suite, iStable, PolyPhen2, SNAP, and PROVEAN], with a view toassess the magnitude of deleteriousness of the mutation. The pathogenicity analysis reveals that the nsSNP could be deleterious inasmuch as it could affect the functionality of the gene, and interfere with protein function. Molecular dynamics simulation of 60ns was performed using GROMACS to analyse structural change due to a mutation (Arg399Gln) that was never examined before. RMSD, RMSF, hydrogen bonds, radius of gyration and SASA analysis showedthe existence of asignificant difference between the native and the mutant protein. The present study gives astrong indication that the XRCC1 locus deserves serious attention, as it could be a potential candidatecontributing to the etio-pathogenesis of the disease.

Investigating the Inhibitory Effect of Wortmannin in the Hotspot Mutation at Codon 1047 of PIK3CA Kinase Domain: A Molecular Docking and Molecular Dynamics Approach

Oncogenic mutations in phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) are the most frequently reported in association with various forms of cancer. Several studies have reported the significance of hotspot mutations in a catalytic subunit of PIK3CA in association with breast cancer. Mutations are frequently observed in the highly conserved region of the kinase domain (797-1068 amino acids) of PIK3CA are activating or gain-of-function mutations. Mutation in codon 1047 occurs in the C-terminal region of the kinase domain with histidine (H) replaced by arginine (R), lysine (L), and tyrosine (Y). Pathogenicity and protein stability predictors PhD-SNP, Align GVGD, HANSA, iStable, and MUpro classified H1047R as highly deleterious when compared to H1047L and H1047Y. To explore the inhibitory activity of Wortmannin toward PIK3CA, the three-dimensional structure of the mutant protein was determined using homology modeling followed by molecular docking and molecular dynamics analysis. Docking studies were performed for the three mutants and native with Wortmannin to measure the differences in their binding pattern. Comparative docking study revealed that H1047R-Wortmannin complex has a higher number of hydrogen bonds as well as the best binding affinity next to the native protein. Furthermore, 100 ns molecular dynamics simulation was initiated with the docked complexes to understand the various changes induced by the mutation. Though Wortmannin was found to nullify the effect of H1047R over the protein, further studies are required for designing a better compound. As SNPs are major genetic variations observed in disease condition, personalized medicine would provide enhanced drug therapy.

Computational Approaches and Resources in Single Amino Acid Substitutions Analysis Toward Clinical Research

Single amino acid substitutions (SAPs) belong to a class of SNPs in the coding region, which alter the protein function during the translation process. Storage of more information regarding SAPs in public databases will soon become a major hurdle in characterizing the functional SAPs. In such a demanding era, biology has to rely on bioinformatics, which can work its way through to solve the problems at hand by cutting huge amount of time and resources that are otherwise wasted. Here, we describe an overview of the existing repositories of variant databases and computational methods in predicting the effects of functional SAPs on protein stability, structure, function, drug response, and protein dynamics. This chapter will inspire many biologists with a greater promise in identifying the functional SAPs at the structural level, thereby understanding the molecular effects that are critical for personalized medicine diagnosis, prognosis, and treatment for diseases.

Application of Evolutionary Based in Silico Methods to Predict the Impact of Single Amino Acid Substitutions in Vitelliform Macular Dystrophy

Recent developments in high-throughput discovery and genotyping have generated a tremendous amount of information about the existence of single amino acid polymorphisms (SAPs). Detailed understanding of the SAPs that affect protein structure and function can provide us valuable insight into disease genotype-phenotype correlations. Functional variants of biological importance are likely to be missed in large-scale analysis. Over the past decade, numerous efforts are underway in understanding and characterizing the potential consequences of variants in assessing the risk associated with vitelliform macular dystrophy (VMD). Yet, in spite of this success, we conducted a first SAP analysis via evolutionary-based in silico pipeline to unravel functional SAPs from a pool, containing both functional and neutral ones. Furthermore, based on the prediction scores, a ranking system was developed to prioritize the functional SAPs in order to minimize the number of SAPs screened for further genotyping.

A Molecular Docking and Dynamics Approach to Screen Potent Inhibitors Against Fosfomycin Resistant Enzyme in Clinical Klebsiella pneumoniae : IDENTIFICATION OF POTENT INHIBITORS AGAINST FosA VARIANT-7

Klebsiella pneumoniae, BA6753 was cultured from a patient in the Clinical Microbiology Laboratory of Christian Medical College. K. pneumoniae, BA6753 has a multidrug resistance plasmid encoding novel FosA variant‐7, fosfomycin resistance enzyme. Minimal side effects and a wide range of bactericidal activity of fosfomycin have resulted in its expanded clinical use that prompts the rise of fosfomycin‐resistant strains. At present, there are no effective inhibitors available to conflict the FosA‐medicated fosfomycin resistance. To develop effective FosA inhibitors, it is crucial to understand the structural and dynamic properties of resistance enzymes. Hence, the present study focuses on the identification of potent inhibitors that can effectively bind to the fosfomycin resistance enzyme, thus predispose the target to inactivate by the second antibiotic. Initially, a series of active compounds were screened against the resistant enzyme, and the binding affinities were confirmed using docking simulation analysis. For efficient activity, the binding affinity of the resistance enzyme ought to be high with the inhibitor than the fosfomycin drug. Consequently, the enzyme‐ligand complex which showed higher binding affinity than the fosfomycin was employed for subsequent analysis. The stability of the top scoring enzyme‐ligand complex was further validated using molecular dynamics simulation studies. On the whole, we presume that the compound 19583672 demonstrates a higher binding affinity for the resistance enzyme comparing to other compounds and fosfomycin. We believe that further enhancement of the lead compound can serve as a potential inhibitor against resistance enzyme in drug discovery process. J. Cell. Biochem. 118: 4088–4094, 2017.