Interdisciplinary in silico studies to understand in-depth molecular level mechanism of drug resistance involving NS3-4A protease of HCV.

Hepatitis C virus (HCV) causes hepatitis, a life-threatening disease responsible for liver cirrhosis. Urgent measures have been taken to develop therapeutics against this deadly pathogen. NS3/4A protease is an extremely important target. A series of inhibitors have been developed against this viral protease including Faldaprevir. Unfortunately, the error-prone viral RNA polymerase causes the emergence of resistance, thereby causing reduced effectiveness of those peptidomimetic inhibitors. Among the drug resistant variants, three single amino acid residues (R155, A156 and D168) are notable for their presence in clinical isolates and also their effectivity against most of the known inhibitors in clinical development. Therefore, it is crucial to understand the mechanistic role of those drug resistant variants while designing potent novel inhibitors. In this communication, we have deeply analyzed through using in silico studies to understand the molecular mechanism of alteration of inhibitor binding between wild type and its R155K, A156V and D168V variants. Principal component analysis was carried to identify the backbone fluctuations of important residues in HCV NS3/4A responsible for the inhibitor binding and maintaining drug resistance. Free energy landscape as a function of the principal components has been used to identify the stability and conformation of the key residues that regulate inhibitor binding and their impact in developing drug resistance. Our findings are consistent with the trend of experimental results. The observations are also true in case of other Faldaprevir-like peptidomimetic inhibitors. Understanding this binding mechanism would be significant for the development of novel inhibitors with less susceptibility towards drug resistance.Communicated by Ramaswamy H. Sarma.

Characterization of a Class A ß-Lactamase from Francisella tularensis (Ftu-1) Belonging to a Unique Subclass toward Understanding AMR.

ß-lactamase production with vast catalytic divergence in the pathogenic strain limits the antibiotic spectrum in the clinical environment. Class A carbapenemase shares significant sequence similarities, structural features, and common catalytic mechanisms although their resistance spectrum differs from class A ß-lactamase in carbapenem and monobactam hydrolysis. In other words, it limited the antibiotic treatment option against infection, causing carbapenemase-producing superbugs. Ftu-1 is a class A ß-lactamase expressed by the Francisella tularensis strain, a potent causative organism of tularemia. The chromosomally encoded class A ß-lactamase shares two conserved cysteine residues, a common characteristic of a carbapenemase, and a distinctive class in the phylogenetic tree. Complete biochemical and biophysical characterization of the enzyme was performed to understand the overall stability and environmental requirements to perform optimally. To comprehend the enzyme-drug interaction and its profile toward various chemistries of ß-lactam and ß-lactamase inhibitors, comprehensive kinetic and thermodynamic analyses were conducted using various ß-lactam drugs. The dynamic property of Ftu-1 ß-lactamase was also predicted using molecular dynamics (MD) simulation to compare its loop flexibility and ligand binding with other related class A ß-lactamases. Overall, this study fosters a comprehensive understanding of Ftu-1, proposed to be an intermediate class by characterizing its kinetic profiling, stability by biochemical and biophysical methodologies, and susceptibility profiling. This understanding would be beneficial for the design of new-generation therapeutics.

In silico study of cox protein from P2 type enteric bacteriophages based on sequence, structure and dynamics to understand its functional integrity.

Cox protein plays a critical role in deciding the lytic-lysogenic switch of P2 enteric phages. This phenomenon makes Cox protein one of the most important candidates in developing novel phage-based therapeutics against antibacterial resistant pathogens. The principle focus concerning protein and its decision making is a DNA binding event, which helps to regulate differential promoter expression. In the current study, we have attempted to understand the sequence, structural and dynamic features associated with Cox protein and its DNA binding. Unavailability of information was a big burden in further proceedings. We have done an extensive literature search to develop a database of Cox with relevant information. That information coupled with the methods of Sequence-based phylogenetic and conservation studies, Homology Modelling, Atomic-level Docking and Molecular Dynamics (MD) Simulation (50 ns each for 10 systems, i.e. total of 500 ns) were performed in the current study. Analysis of those extensive studies has provided us the required sequence to structure to dynamics to functional understanding. Our present study would indeed be very helpful in understanding the biochemical mechanism of Cox activation as well as designing potential phage therapeutics.

N-terminal truncation of VC0395_0300 protein from Vibrio cholerae does not lead to loss of diguanylate cyclase activity.

The bacterial secondary messenger bis-(3',5')-cyclic-dimeric-guanosine monophosphate (c-di-GMP) has been implicated in the pathogenesis of Vibrio cholerae, due to its significant role in regulating the virulence, biofilm formation and motility of the host organism. The VC0395_0300 protein from V. cholerae, possessing a GGEEF sequence has been established as a diguanylate cyclase (DGC) capable of catalyzing the conversion of two GTP molecules to form cyclic-di-GMP. This in turn, plays a crucial role in allowing the organism to adopt a dual lifestyle, thriving both in human and aquatic systems. The difficulty in procuring sufficient amounts of homogenous soluble protein for structural assessment of the GGDEF domain in VC0395_0300 and the lack of soluble protein yield, prompted the truncation into smaller constructs (Sebox31 and Sebox32) carrying the GGDEF domain. The truncates retained their diguanylate cyclase activity comparable to the wild type, and were able to form biofilms as well. Fluorescence and circular dichroism spectroscopy measurements revealed that the basic structural elements do not show significant changes in the truncated proteins as compared to the full-length. This has also been confirmed using homology modeling and molecular docking of the wild type and truncates. This led us to conclude that the truncated constructs retain their activity in spite of the deletions in the N terminal region. This is supportive of the fact that DGC activity in GGDEF proteins is predominantly dependent on the presence of the conserved GGD(/E)EF domain and its interaction with GTP.

PI3Kα-regulated gelsolin activity is a critical determinant of cardiac cytoskeletal remodeling and heart disease.

Biomechanical stress and cytoskeletal remodeling are key determinants of cellular homeostasis and tissue responses to mechanical stimuli and injury. Here we document the increased activity of gelsolin, an actin filament severing and capping protein, in failing human hearts. Deletion of gelsolin prevents biomechanical stress-induced adverse cytoskeletal remodeling and heart failure in mice. We show that phosphatidylinositol (3,4,5)-triphosphate (PIP3) lipid suppresses gelsolin actin-severing and capping activities. Accordingly, loss of PI3Kα, the key PIP3-producing enzyme in the heart, increases gelsolin-mediated actin-severing activities in the myocardium in vivo, resulting in dilated cardiomyopathy in response to pressure-overload. Mechanical stretching of adult PI3Kα-deficient cardiomyocytes disrupts the actin cytoskeleton, which is prevented by reconstituting cells with PIP3. The actin severing and capping activities of recombinant gelsolin are effectively suppressed by PIP3. Our data identify the role of gelsolin-driven cytoskeletal remodeling in heart failure in which PI3Kα/PIP3 act as negative regulators of gelsolin activity.

Putative protein VC0395_0300 from Vibrio cholerae is a diguanylate cyclase with a role in biofilm formation.

The hallmark of the lifecycle of Vibrio cholerae is its ability to switch between two lifestyles - the sessile, non-pathogenic form and the motile, infectious form in human hosts. One of these changes is in the formation of surface biofilms, when in sessile aquatic habitats. The cell-cell interactions within a V. cholerae biofilm are stabilized by the production of an exopolysachharide (EPS) matrix, which in turn is regulated by the ubiquitous secondary messenger, cyclic di-GMP (c-di-GMP), synthesized by proteins containing GGD(/E)EF domains in all prokaryotic systems. Here, we report the functional role of the VC0395_0300 protein (Sebox3) encoded by the chromosome I of V. cholerae, with a GGEEF signature sequence, in the formation of surface biofilms. In our study, we have shown that Escherichia coli containing the full-length Sebox3 displays enhanced biofilm forming ability with cellulose production as quantified and visualized by multiple assays, most notably using FEG-SEM. This has also been corroborated with the lack of motility of host containing Sebox3 in semi-solid media. Searching for the reasons for this biofilm formation, we have demonstrated in vitro that Sebox3 can synthesize c-di-GMP from GTP. The homology derived model of Sebox3 displayed significant conservation of the GGD(/E)EF architecture as well. Hence, we propose that the putative protein VC0395_0300 from V. cholerae is a diguanylate cyclase which has an active role in biofilm formation.

Effect of site-directed mutagenesis at the GGEEF domain of the biofilm forming GGEEF protein from Vibrio cholerae.

Vibrio cholerae, the cause of seven noted pandemics, leads a dual lifecycle-one in the human host in its virulent form, and the other as a sessile, non-virulent bacterium in aquatic bodies in surface biofilms. Surface biofilms have been attributed to be associated with a ubiquitous protein domain present in all branches of bacteria, known as the GGD(/E)EF domain. While the diguanlyate cyclase activities of these proteins are universally established, the role of these proteins as diguanlyate-specific phosphodiesterases in conjunction with a EAL domain has also been reported. The VC0395_0300 protein from V. cholerae which shows biofilm forming abilities also acts as a phosphodiesterase. Interestingly, this GGD(/E)EF protein contains a EAL site in the reverse orientation. We attempted to mutate the GGEEF signature along the sequence by site-directed mutagenesis. The resultant mutants (Sebox5-7) did not show much difference in phosphodiesterase activity in comparison with the wild type protein (Sebox3), indicating the independence of the phosphodiesterase activity of the protein from the GGD(/E)EF domain. However, the ability of the mutants to form surface biofilm was significantly lesser in the case of mutations in the three central positions of the signature domain.

In Silico Designing of an Industrially Sustainable Carbonic Anhydrase Using Molecular Dynamics Simulation.

Carbonic anhydrase (CA) is a family of metalloenzymes that has the potential to sequestrate carbon dioxide (CO2) from the environment and reduce pollution. The goal of this study is to apply protein engineering to develop a modified CA enzyme that has both higher stability and activity and hence could be used for industrial purposes. In the current study, we have developed an in silico method to understand the molecular basis behind the stability of CA. We have performed comparative molecular dynamics simulation of two homologous α-CA, one of thermophilic origin (Sulfurihydrogenibium sp.) and its mesophilic counterpart (Neisseria gonorrhoeae), for 100 ns each at 300, 350, 400, and 500 K. Comparing the trajectories of two proteins using different stability-determining factors, we have designed a highly thermostable version of mesophilic α-CA by introducing three mutations (S44R, S139E, and K168R). The designed mutant α-CA maintains conformational stability at high temperatures. This study shows the potential to develop industrially stable variants of enzymes while maintaining high activity.

Comparative molecular dynamics simulation studies for determining factors contributing to the thermostability of chemotaxis protein "CheY".

Comparative molecular dynamics simulations of chemotaxis protein "CheY" from thermophilic origin Thermotoga maritima and its mesophilic counterpart Salmonella enterica have been performed for 10 ns each at 300 and 350 K, and 20 ns each at 400 and 450 K. The trajectories were analyzed in terms of different factors like root-mean-square deviation, root-mean-square fluctuation, radius of gyration, solvent accessible surface area, H-bonds, salt bridge content, and protein-solvent interactions which indicate distinct differences between the two of them. The two proteins also follow dissimilar unfolding pathways. The overall flexibility calculated by the trace of the diagonalized covariance matrix displays similar flexibility of both the proteins near their optimum growth temperatures. However, at higher temperatures mesophilic protein shows increased overall flexibility than its thermophilic counterpart. Principal component analysis also indicates that the essential subspaces explored by the simulations of two proteins at different temperatures are nonoverlapping and they show significantly different directions of motion. However, there are significant overlaps within the trajectories and similar direction of motions are observed for both proteins at 300 K. Overall, the mesophilic protein leads to increased conformational sampling of the phase space than its thermophilic counterpart. This is the first ever study of thermostability of CheY protein homologs by using protein dynamism as a main impact. Our study might be used as a model for studying the molecular basis of thermostability of two homologous proteins from two organisms living at different temperatures with less visible differences.