Cytochrome c (CytC), a one-electron carrier, transfers electrons from complex bc1 to cytochrome c oxidase (CcO) in the electron-transport chain. Electrostatic interaction with the partners, complex bc1 and CcO, is ensured by a lysine cluster near the heme forming the Universal Binding Site (UBS). We constructed three mutant variants of mitochondrial CytC with one (2Mut), four (5Mut), and five (8Mut) Lys->Glu substitutions in the UBS and some compensating Glu->Lys substitutions at the periphery of the UBS for charge compensation. All mutants showed a 4-6 times increased peroxidase activity and accelerated binding of cyanide to the ferric heme of CytC. In contrast, decomposition of the cyanide complex with ferrous CytC, as monitored by magnetic circular dichroism spectroscopy, was slower in mutants compared to WT. Molecular dynamic simulations revealed the increase in the fluctuations of Cα atoms of individual residues of mutant CytC compared to WT, especially in the Ω-loop (70-85), which can cause destabilization of the Fe…S(Met80) coordination link, facilitation of the binding of exogenous ligands cyanide and peroxide, and an increase in peroxidase activity. It was found that only one substitution K72E is enough to induce all these changes, indicating the significance of K72 and the Ω-loop (70-85) for the structure and physiology of mitochondrial CytC. In this work, we also propose using a ferro-ferricyanide buffer as a substrate to monitor the peroxidase activity of CytC. This new approach allows us to determine the rate of peroxidase activity at moderate (200 µM) concentrations of H2O2 and avoid complications of radical formation during the reaction.

SHP2 belongs to a cytoplasmic non-receptor protein tyrosine phosphatase class. It plays a critical role in the development of various cancers, such as gastric cancer, leukemia, and breast cancer. Thus, SHP2 has gained the interest of researchers as a potential target for inhibiting tumor cell proliferation in SHP2-dependent cancers. This study employed pharmacophore-based virtual screening, molecular docking, molecular dynamic (MD) simulations, MM/PBSA, and principal component analysis (PCA), followed by ADME prediction. We selected three potential hits from a collective database of more than one million chemical compounds. The stability of these selected hit-protein complexes was analyzed using 500 ns MD simulations and binding free energy calculations. The identified hits Lig_1, Lig_6, and Lig_14 demonstrated binding free energies of -161.49 kJ/mol, -151.28 kJ/mol, and -107.13 kJ/mol, respectively, compared to the reference molecule (SHP099) with a ΔG of -71.48 kJ/mol. Our results showed that the identified compounds could be used as promising candidates for selective SHP2 allosteric inhibition in cancer.

Tumor suppressor cylindromatosis protein (CYLD) regulates NF-κB and JNK signaling pathways by cleaving K63-linked poly-ubiquitin chain from its substrate molecules and thus preventing the progression of tumorigenesis and metastasis of the cancer cells. Mutations in CYLD can cause aberrant structure and abnormal functionality leading to tumor formation. In this study, we utilized several computational tools such as PANTHER, PROVEAN, PredictSNP, PolyPhen-2, PhD-SNP, PON-P2, and SIFT to find out deleterious nsSNPs. We also highlighted the damaging impact of those deleterious nsSNPs on the structure and function of the CYLD utilizing ConSurf, I-Mutant, SDM, Phyre2, HOPE, Swiss-PdbViewer, and Mutation 3D. We shortlisted 18 high-risk nsSNPs from a total of 446 nsSNPs recorded in the NCBI database. Based on the conservation profile, stability status, and structural impact analysis, we finalized 13 nsSNPs. Molecular docking analysis and molecular dynamic simulation concluded the study with the findings of two significant nsSNPs (R830K, H827R) which have a remarkable impact on binding affinity, RMSD, RMSF, radius of gyration, and hydrogen bond formation during CYLD-ubiquitin interaction. The principal component analysis compared native and two mutants R830K and H827R of CYLD that signify structural and energy profile fluctuations during molecular dynamic (MD) simulation. Finally, the protein-protein interaction network showed CYLD interacts with 20 proteins involved in several biological pathways that mutations can impair. Considering all these in silico analyses, our study recommended conducting large-scale association studies of nsSNPs of CYLD with cancer as well as designing precise medications against diseases associated with these polymorphisms.

A cell\'s ability to survive and to evade cancer is contingent on its ability to retain genomic integrity, which can be seriously compromised when nucleic acid phosphodiester bonds are disrupted. DNA Ligase 1 (LIG1) plays a key role in genome maintenance by sealing single-stranded nicks that are produced during DNA replication and repair. Autosomal recessive mutations in a limited number of individuals have been previously described for this gene. Here we report a homozygous LIG1 mutation (p.A624T), affecting a universally conserved residue, in a patient presenting with leukopenia, neutropenia, lymphopenia, pan-hypogammaglobulinemia, and diminished in vitro response to mitogen stimulation. Patient fibroblasts expressed normal levels of LIG1 protein but exhibited impaired growth, poor viability, high baseline levels of gamma-H2AX foci, and an enhanced susceptibility to DNA-damaging agents. The mutation reduced LIG1 activity by lowering its affinity for magnesium 2.5-fold. Remarkably, it also increased LIG1 fidelity > 50-fold against 3\' end 8-Oxoguanine mismatches, exhibiting a marked reduction in its ability to process such nicks. This is expected to yield increased ss- and dsDNA breaks. Molecular dynamic simulations, and Residue Interaction Network studies, predicted an allosteric effect for this mutation on the protein loops associated with the LIG1 high-fidelity magnesium, as well as on DNA binding within the adenylation domain. These dual alterations of suppressed activity and enhanced fidelity, arising from a single mutation, underscore the mechanistic picture of how a LIG1 defect can lead to severe immunological disease.

Due to the low temperature, the Antarctic marine environment is challenging for protein functioning. Cold-adapted organisms have evolved proteins endowed with higher flexibility and lower stability in comparison to their thermophilic homologs, resulting in enhanced reaction rates at low temperatures. The Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125) genome is one of the few examples of coexistence of multiple hemoglobin genes encoding, among others, two constitutively transcribed 2/2 hemoglobins (2/2Hbs), also named truncated Hbs (TrHbs), belonging to the Group II (or O), annotated as PSHAa0030 and PSHAa2217. In this work, we describe the ligand binding kinetics and their interrelationship with the dynamical properties of globin Ph-2/2HbO-2217 by combining experimental and computational approaches and implementing a new computational method to retrieve information from molecular dynamic trajectories. We show that our approach allows us to identify docking sites within the protein matrix that are potentially able to transiently accommodate ligands and migration pathways connecting them. Consistently with ligand rebinding studies, our modeling suggests that the distal heme pocket is connected to the solvent through a low energy barrier, while inner cavities play only a minor role in modulating rebinding kinetics.

Introduction: The most common primary brain tumor in adults is glioblastoma multiforme (GBM), accounting for 45.2% of all cases. The characteristics of GBM, a highly aggressive brain tumor, include rapid cell division and a propensity for necrosis. Regretfully, the prognosis is extremely poor, with only 5.5% of patients surviving after diagnosis. Methodology: To eradicate these kinds of complicated diseases, significant focus is placed on developing more effective drugs and pinpointing precise pharmacological targets. Finding appropriate biomarkers for drug discovery entails considering a variety of factors, including illness states, gene expression levels, and interactions between proteins. Using statistical techniques like p-values and false discovery rates, we identified differentially expressed genes (DEGs) as the first step in our research for identifying promising biomarkers in GBM. Of the 132 genes, 13 showed upregulation, and only 29 showed unique downregulation. No statistically significant changes in the expression of the remaining genes were observed. Results: Matrix metallopeptidase 9 (MMP9) had the greatest degree in the hub biomarker gene identification, followed by (periostin (POSTN) at 11 and Hes family BHLH transcription factor 5 (HES5) at 9. The significance of the identification of each hub biomarker gene in the initiation and advancement of glioblastoma multiforme was brought to light by the survival analysis. Many of these genes participate in signaling networks and function in extracellular areas, as demonstrated by the enrichment analysis.We also identified the transcription factors and kinases that control proteins in the proteinprotein interactions (PPIs) of the DEGs. Discussion: We discovered drugs connected to every hub biomarker. It is an appealing therapeutic target for inhibiting MMP9 involved in GBM. Molecular docking investigations indicated that the chosen complexes (carmustine, lomustine, marimastat, and temozolomide) had high binding affinities of -6.3, -7.4, -7.7, and -8.7 kcal/mol, respectively, the mean root-mean-square deviation (RMSD) value for the carmustine complex and marimastat complex was 4.2 Å and 4.9 Å, respectively, and the lomustine and temozolomide complex system showed an average RMSD of 1.2 Å and 1.6 Å, respectively. Additionally, high stability in root-mean-square fluctuation (RMSF) analysis was observed with no structural conformational changes among the atomic molecules. Thus, these in silico investigations develop a new way for experimentalists to target lethal diseases in future.

We provide promising computational (in silico) data on phytochemicals (compounds 1-10) from Arabian Peninsula medicinal plants as strong binders, targeting 3-chymotrypsin-like protease (3CLPro) and papain-like proteases (PLPro) of SARS-CoV-2. Compounds 1-10 followed the Lipinski rules of five (RO5) and ADMET analysis, exhibiting drug-like characters. Non-covalent (reversible) docking of compounds 1-10 demonstrated their binding with the catalytic dyad (CYS145 and HIS41) of 3CLPro and catalytic triad (CYS111, HIS272, and ASP286) of PLPro. Moreover, the implementation of the covalent (irreversible) docking protocol revealed that only compounds 7, 8, and 9 possess covalent warheads, which allowed the formation of the covalent bond with the catalytic dyad (CYS145) in 3CLPro and the catalytic triad (CYS111) in PLPro. Root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), and radius of gyration (Rg) analysis from molecular dynamic (MD) simulations revealed that complexation between ligands (compounds 7, 8, and 9) and 3CLPro and PLPro was stable, and there was less deviation of ligands. Overall, the in silico data on the inherent properties of the above phytochemicals unravel the fact that they can act as reversible inhibitors for 3CLPro and PLPro. Moreover, compounds 7, 8, and 9 also showed their novel properties to inhibit dual targets by irreversible inhibition, indicating their effectiveness for possibly developing future drugs against SARS-CoV-2. Nonetheless, to confirm the theoretical findings here, the effectiveness of the above compounds as inhibitors of 3CLPro and PLPro warrants future investigations using suitable in vitro and in vivo tests.

Chaperonins Hsp60s are required for cellular vitality by assisting protein folding in an ATP-dependent mechanism. Although conserved, the human mitochondrial mHsp60 exhibits molecular characteristics distinct from the E. coli GroEL, with different conformational assembly and higher subunit association dynamics, suggesting a different mechanism. We previously found that the pathological mutant mHsp60V72I exhibits enhanced subunit association stability and ATPase activity. To provide structural explanations for the V72I mutational effects, here we determined a cryo-EM structure of mHsp60V72I. Our structural analysis combined with molecular dynamic simulations showed mHsp60V72I with increased inter-subunit interface, binding free energy, and dissociation force, all contributing to its enhanced subunit association stability. The gate to the nucleotide-binding (NB) site in mHsp60V72I mimicked the open conformation in the nucleotide-bound state with an additional open channel leading to the NB site, both promoting the mutant\'s ATPase activity. Our studies highlight the importance of mHsp60\'s characteristics in its biological function.

BACKGROUND: Single nucleotide polymorphisms (SNPs) have been linked with prostate cancer (PCa) and have shown potential as prognostic markers for advanced stages. Loss of function mutations in PKCι have been linked with increased risk of malignancy by enhancing tumor cell motility and invasion. We have evaluated the impact of two coding region SNPs on the PKCι gene (PRKCI) and their prognostic potential.
METHODS: Genotypic association of non-synonymous PKCι SNPs rs1197750201 and rs1199520604 with PCa was determined through tetra-ARMS PCR. PKCι was docked with interacting partner Par-6 to determine the effect of these variants on PKCι binding capabilities. Molecular dynamic simulations of PKCι docked with Par-6 were performed to determine variant effects on PKCι protein interactions. The possible impact of changes in PKCι protein interactions on epithelial cell polarity was hypothesized.
RESULTS: PKCι rs1199520604 mutant genotype TT showed association with PCa (p = 0.0055), while rs1197750201 mutant genotype AA also showed significant association with PCa (P = 0.0006). The binding interaction of PKCι with Par-6 was altered for both variants, with changes in Van der Waals energy and electrostatic energy of docked structures.
CONCLUSIONS: Genotypic analysis of two non-synonymous PKCι variants in association with PCa prognosis was performed. Both variants in the PB1 domain showed potential as a prognostic marker for PCa. In silico analysis of the effect of the variants on PKCι protein interactions indicated they may be involved in PCa progression through aberration of epithelial cell polarity pathways.

One of the most common primary resistance mechanism of multi-drug resistant (MDR) Gram negative pathogenic bacteria to combat β-lactam antibiotics, such as penicillins, cephalosporins and carbapenems is the generation of β- lactamases. The uropathogenic E. coli is mostly getting multi-drug resistance due to the synthesis of AmpC β-lactamases and therefore new antibiotics and inhibitors are needed to treat the evolving infections. The current study was designed for targetting AmpC β-lactamase of E. coli using molecular docking based virtual screening, linking fragments for designing novel compounds and binding mode analysis using molecular dynamic simulation with target protein. The FCH group all-purpose fragment library consisting of 9388 fragments has been screened against AmpC β-lactamase protein of E. coli and the antibiotics and anti-infectives used in treatment of Urinary tract Infections (UTIs) were also screened with AmpC β-lactamase protein. Among the 9388 fragments, 339 fragment candidates were selected and linked with cefepime antibiotic having maximum binding affinity for AmpC target protein. Computational analysis of interactions as well as molecular dynamics (MD) simulations were also conducted for identifying the most promising ligand-pocket complexes from docking investigations to comprehend their thermodynamic properties and verify the docking outcomes as well. Overall, the linked complexes (LCs) showed good binding interactions with AmpC β-lactamase. Interestingly, our fragment-based LCs remained relatively stable in comparison with cefepime antibiotic. Moreover, S12 fragment linked complex remained the most stable during 50 ns with remarkable number of interactions indicating it as promising candidate in novel lead discovery against MDR E. coli infections.