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Journal of Biomolecular Structure &... Feb 20223CL is the main protease of the novel coronavirus (SARS-CoV-2) responsible for their intracellular duplication. Based on virtual screening technology and molecular...
3CL is the main protease of the novel coronavirus (SARS-CoV-2) responsible for their intracellular duplication. Based on virtual screening technology and molecular dynamics simulation, we found 23 approved clinical drugs such as Viomycin, Capastat, Carfilzomib and Saquinavir, which showed high affinity with the 3CL active sites. These findings showed that there were potential drugs that inhibit SARS-Cov-2's 3CL in the current clinical drug library, and these drugs can be further tested or chemically modified for the treatment of COVID-19.Communicated by Ramaswamy H. Sarma.
Topics: COVID-19; Humans; Molecular Docking Simulation; Peptide Hydrolases; Pharmaceutical Preparations; Protease Inhibitors; SARS-CoV-2
PubMed: 32909528
DOI: 10.1080/07391102.2020.1817786 -
Frontiers in Chemistry 2022CmnC is an α-ketoglutarate (α-KG)-dependent non-heme iron oxygenase involved in the formation of the l-capreomycidine (l-Cap) moiety in capreomycin (CMN) biosynthesis....
Crystal structure of the α-ketoglutarate-dependent non-heme iron oxygenase CmnC in capreomycin biosynthesis and its engineering to catalyze hydroxylation of the substrate enantiomer.
CmnC is an α-ketoglutarate (α-KG)-dependent non-heme iron oxygenase involved in the formation of the l-capreomycidine (l-Cap) moiety in capreomycin (CMN) biosynthesis. CmnC and its homologues, VioC in viomycin (VIO) biosynthesis and OrfP in streptothricin (STT) biosynthesis, catalyze hydroxylation of l-Arg to form β-hydroxy l-Arg (CmnC and VioC) or β,γ-dihydroxy l-Arg (OrfP). In this study, a combination of biochemical characterization and structural determination was performed to understand the substrate binding environment and substrate specificity of CmnC. Interestingly, despite having a high conservation of the substrate binding environment among CmnC, VioC, and OrfP, only OrfP can hydroxylate the substrate enantiomer d-Arg. Superposition of the structures of CmnC, VioC, and OrfP revealed a similar folds and overall structures. The active site residues of CmnC, VioC, and OrfP are almost conserved; however Leu136, Ser138, and Asp249 around the substrate binding pocket in CmnC are replaced by Gln, Gly, and Tyr in OrfP, respectively. These residues may play important roles for the substrate binding. The mutagenesis analysis revealed that the triple mutant CmnC switches the substrate stereoselectivity from l-Arg to d-Arg with ∼6% relative activity. The crystal structure of CmnC in complex with d-Arg revealed that the substrate loses partial interactions and adopts a different orientation in the binding site. This study provides insights into the enzyme engineering to α-KG non-heme iron oxygenases for adjustment to the substrate stereoselectivity and development of biocatalysts.
PubMed: 36176888
DOI: 10.3389/fchem.2022.1001311 -
Journal of Molecular Biology Jan 2015Previous structural studies suggested that ribosomal translocation is accompanied by large interdomain rearrangements of elongation factor G (EF-G). Here, we follow the...
Previous structural studies suggested that ribosomal translocation is accompanied by large interdomain rearrangements of elongation factor G (EF-G). Here, we follow the movement of domain IV of EF-G relative to domain II of EF-G using ensemble and single-molecule Förster resonance energy transfer. Our results indicate that ribosome-free EF-G predominantly adopts a compact conformation that can also, albeit infrequently, transition into a more extended conformation in which domain IV moves away from domain II. By contrast, ribosome-bound EF-G predominantly adopts an extended conformation regardless of whether it is interacting with pretranslocation ribosomes or with posttranslocation ribosomes. Our data suggest that ribosome-bound EF-G may also occasionally sample at least one more compact conformation. GTP hydrolysis catalyzed by EF-G does not affect the relative stability of the observed conformations in ribosome-free and ribosome-bound EF-G. Our data support a model suggesting that, upon binding to a pretranslocation ribosome, EF-G moves from a compact to a more extended conformation. This transition is not coupled to but likely precedes both GTP hydrolysis and mRNA/tRNA translocation.
Topics: Escherichia coli; Gene Rearrangement; Peptide Elongation Factor G; Protein Conformation; RNA, Messenger; RNA, Transfer; Ribosomes; Sequence Analysis, DNA; Translocation, Genetic
PubMed: 25463439
DOI: 10.1016/j.jmb.2014.11.010 -
ELife Jun 2019Applying pre-steady state kinetics to an based reconstituted translation system, we have studied how the antibiotic viomycin affects the accuracy of genetic code...
Applying pre-steady state kinetics to an based reconstituted translation system, we have studied how the antibiotic viomycin affects the accuracy of genetic code reading. We find that viomycin binds to translating ribosomes associated with a ternary complex (TC) consisting of elongation factor Tu (EF-Tu), aminoacyl tRNA and GTP, and locks the otherwise dynamically flipping monitoring bases A1492 and A1493 into their active conformation. This effectively prevents dissociation of near- and non-cognate TCs from the ribosome, thereby enhancing errors in initial selection. Moreover, viomycin shuts down proofreading-based error correction. Our results imply a mechanism in which the accuracy of initial selection is achieved by larger backward rate constants toward TC dissociation rather than by a smaller rate constant for GTP hydrolysis for near- and non-cognate TCs. Additionally, our results demonstrate that translocation inhibition, rather than error induction, is the major cause of cell growth inhibition by viomycin.
Topics: Anti-Bacterial Agents; Cell-Free System; Protein Biosynthesis; Protein Synthesis Inhibitors; Viomycin
PubMed: 31172942
DOI: 10.7554/eLife.46124 -
Proceedings of the National Academy of... Oct 2014Translocation of mRNA and tRNAs through the ribosome is catalyzed by a universally conserved elongation factor (EF-G in prokaryotes and EF-2 in eukaryotes). Previous...
Translocation of mRNA and tRNAs through the ribosome is catalyzed by a universally conserved elongation factor (EF-G in prokaryotes and EF-2 in eukaryotes). Previous studies have suggested that ribosome-bound EF-G undergoes significant structural rearrangements. Here, we follow the movement of domain IV of EF-G, which is critical for the catalysis of translocation, relative to protein S12 of the small ribosomal subunit using single-molecule FRET. We show that ribosome-bound EF-G adopts distinct conformations corresponding to the pre- and posttranslocation states of the ribosome. Our results suggest that, upon ribosomal translocation, domain IV of EF-G moves toward the A site of the small ribosomal subunit and facilitates the movement of peptidyl-tRNA from the A to the P site. We found no evidence of direct coupling between the observed movement of domain IV of EF-G and GTP hydrolysis. In addition, our results suggest that the pretranslocation conformation of the EF-G-ribosome complex is significantly less stable than the posttranslocation conformation. Hence, the structural rearrangement of EF-G makes a considerable energetic contribution to promoting tRNA translocation.
Topics: Biological Transport; Catalysis; Fluorescence Resonance Energy Transfer; Guanosine Triphosphate; Microscopy; Peptide Elongation Factor G; Protein Binding; Protein Structure, Tertiary; Protein Synthesis Inhibitors; Protein Transport; RNA, Messenger; RNA, Transfer; Ribosomes; Viomycin
PubMed: 25288752
DOI: 10.1073/pnas.1410873111 -
Journal of the American Chemical Society Jun 2018Hydroxylation of aliphatic carbons by nonheme Fe(IV)-oxo (ferryl) complexes proceeds by hydrogen-atom (H•) transfer (HAT) to the ferryl and subsequent coupling between...
Hydroxylation of aliphatic carbons by nonheme Fe(IV)-oxo (ferryl) complexes proceeds by hydrogen-atom (H•) transfer (HAT) to the ferryl and subsequent coupling between the carbon radical and Fe(III)-coordinated oxygen (termed rebound). Enzymes that use H•-abstracting ferryl complexes for other transformations must either suppress rebound or further process hydroxylated intermediates. For olefin-installing C-C desaturations, it has been proposed that a second HAT to the Fe(III)-OH complex from the carbon α to the radical preempts rebound. Deuterium (H) at the second site should slow this step, potentially making rebound competitive. Desaturations mediated by two related l-arginine-modifying iron(II)- and 2-(oxo)glutarate-dependent (Fe/2OG) oxygenases behave oppositely in this key test, implicating different mechanisms. NapI, the l-Arg 4,5-desaturase from the naphthyridinomycin biosynthetic pathway, abstracts H• first from C5 but hydroxylates this site (leading to guanidine release) to the same modest extent whether C4 harbors H or H. By contrast, an unexpected 3,4-desaturation of l-homoarginine (l-hArg) by VioC, the l-Arg 3-hydroxylase from the viomycin biosynthetic pathway, is markedly disfavored relative to C4 hydroxylation when C3 (the second hydrogen donor) harbors H. Anchimeric assistance by N6 permits removal of the C4-H as a proton in the NapI reaction, but, with no such assistance possible in the VioC desaturation, a second HAT step (from C3) is required. The close proximity (≤3.5 Å) of both l-hArg carbons to the oxygen ligand in an X-ray crystal structure of VioC harboring a vanadium-based ferryl mimic supports and rationalizes the sequential-HAT mechanism. The results suggest that, although the sequential-HAT mechanism is feasible, its geometric requirements may make competing hydroxylation unavoidable, thus explaining the presence of α-heteroatoms in nearly all native substrates for Fe/2OG desaturases.
Topics: Binding Sites; Deuterium; Homoarginine; Hydroxylation; Iron; Ketoglutaric Acids; Kinetics; Mixed Function Oxygenases; Models, Chemical; Oxidation-Reduction; Stereoisomerism
PubMed: 29708749
DOI: 10.1021/jacs.8b01933 -
ACS Omega Apr 2024Colorectal cancer (CRC) has witnessed a concerning increase in incidence and poses a significant therapeutic challenge due to its poor prognosis. There is a pressing...
Colorectal cancer (CRC) has witnessed a concerning increase in incidence and poses a significant therapeutic challenge due to its poor prognosis. There is a pressing demand to identify novel drug therapies to combat CRC. In this study, we addressed this need by utilizing the pharmacological profiles of anticancer drugs from the Genomics of Drug Sensitivity in Cancer (GDSC) database and developed QSAR models using the Support Vector Machine (SVM) algorithm for prediction of alternative and promiscuous anticancer compounds for CRC treatment. Our QSAR models demonstrated their robustness by achieving a high correlation of determination () after 10-fold cross-validation. For 12 CRC cell lines, ranged from 0.609 to 0.827. The highest performance was achieved for SW1417 and GP5d cell lines with values of 0.827 and 0.786, respectively. Further, we listed the most common chemical descriptors in the drug profiles of the CRC cell lines and we also further reported the correlation of these descriptors with drug activity. The KRFP314 fingerprint was the predominantly occurring descriptor, with the KRFPC314 fingerprint following closely in prevalence within the drug profiles of the CRC cell lines. Beyond predictive modeling, we also confirmed the applicability of our developed QSAR models via methods by conducting descriptor-drug analyses and recapitulating drug-to-oncogene relationships. We also identified two potential anti-CRC FDA-approved drugs, viomycin and diamorphine, using QSAR models. To ensure the easy accessibility and utility of our research findings, we have incorporated these models into a user-friendly prediction Web server named "ColoRecPred", available at https://project.iith.ac.in/cgntlab/colorecpred. We anticipate that this Web server can be used for screening of chemical libraries to identify potential anti-CRC drugs.
PubMed: 38680332
DOI: 10.1021/acsomega.4c01195 -
RNA (New York, N.Y.) Jan 2016Here we report that the specificity of peptide release in the ribosome on a nonstop mRNA by ArfA and RF2 is achieved by an induced-fit mechanism. Using RF2 that is...
Here we report that the specificity of peptide release in the ribosome on a nonstop mRNA by ArfA and RF2 is achieved by an induced-fit mechanism. Using RF2 that is methylated on the glutamine of its GGQ motif (RF2(m)), we show that methylation substantially increases the rate of ArfA/RF2-catalyzed peptide release on a nonstop mRNA that does not occupy the ribosomal A site, but has only a modest effect on k(cat) by the same proteins on longer nonstop mRNAs occupying the A site of the mRNA channel in the ribosome. Our data suggest that enhancement in the kcat of peptide release by ArfA and RF2 under the cognate decoding condition is the result of favorable conformational changes in the nonstop complex. We demonstrate a shared mechanism between canonical and nonstop termination, supported by similarities in the kinetic mechanisms in antibiotic inhibition and methylation-correlated enhancement in the rate of peptide release. Despite these similarities, our data suggest that nonstop termination differs from canonical pathway in the downstream event of recycling.
Topics: Biocatalysis; Escherichia coli; Escherichia coli Proteins; Methylation; Paromomycin; Peptide Chain Termination, Translational; Peptide Termination Factors; Peptides; Protein Biosynthesis; RNA-Binding Proteins; Ribosomes; Viomycin
PubMed: 26554029
DOI: 10.1261/rna.053082.115 -
Nature Communications Sep 2014The antibiotic streptomycin is widely used in the treatment of microbial infections. The primary mechanism of action is inhibition of translation by binding to the...
The antibiotic streptomycin is widely used in the treatment of microbial infections. The primary mechanism of action is inhibition of translation by binding to the ribosome, but how it enters the bacterial cell is unclear. Early in the study of this antibiotic, a mysterious streptomycin-induced potassium efflux preceding any decrease in viability was observed; it was speculated that this changed the electrochemical gradient such that streptomycin better accessed the cytoplasm. Here we use a high-throughput screen to search for compounds targeting the mechanosensitive channel of large conductance (MscL) and find dihydrostreptomycin among the 'hits'. Furthermore, we find that MscL is not only necessary for the previously described streptomycin-induced potassium efflux, but also directly increases MscL activity in electrophysiological studies. The data suggest that gating MscL is a novel mode of action of dihydrostreptomycin, and that MscL's large pore may provide a mechanism for cell entry.
Topics: Anti-Bacterial Agents; Dihydrostreptomycin Sulfate; Escherichia coli; Escherichia coli Proteins; High-Throughput Screening Assays; Ion Channels; Patch-Clamp Techniques; Potassium; Spectinomycin; Streptomycin; Viomycin
PubMed: 25205267
DOI: 10.1038/ncomms5891