scholarly journals Phylogenetic Sequence Variations in Bacterial rRNA Affect Species-Specific Susceptibility to Drugs Targeting Protein Synthesis

2011 ◽  
Vol 55 (9) ◽  
pp. 4096-4102 ◽  
Author(s):  
Subramanian Akshay ◽  
Mihai Bertea ◽  
Sven N. Hobbie ◽  
Björn Oettinghaus ◽  
Dimitri Shcherbakov ◽  
...  
Keyword(s):  
Binding Pocket ◽  
Drug Susceptibility ◽  
Drug Binding ◽  
23S Rrna ◽  
Content Type ◽  
Sequence Variations ◽  
Binding Pockets ◽  
Species Specific ◽  
Peptidyltransferase Center ◽  
A Site

ABSTRACTAntibiotics targeting the bacterial ribosome typically bind to highly conserved rRNA regions with only minor phylogenetic sequence variations. It is unclear whether these sequence variations affect antibiotic susceptibility or resistance development. To address this question, we have investigated the drug binding pockets of aminoglycosides and macrolides/ketolides. The binding site of aminoglycosides is located within helix 44 of the 16S rRNA (A site); macrolides/ketolides bind to domain V of the 23S rRNA (peptidyltransferase center). We have used mutagenesis of rRNA sequences inMycobacterium smegmatisribosomes to reconstruct the different bacterial drug binding sites and to study the effects of rRNA sequence variations on drug activity. Our results provide a rationale for differences in species-specific drug susceptibility patterns and species-specific resistance phenotypes associated with mutational alterations in the drug binding pocket.

scholarly journals In Vitro Susceptibility Testing of GSK656 against Mycobacterium Species

2019 ◽  
Vol 64 (2) ◽  
Author(s):  
Wenzhu Dong ◽  
Shanshan Li ◽  
Shu’an Wen ◽  
Wei Jing ◽  
Jin Shi ◽  
...  
Keyword(s):  
Sequence Similarity ◽  
Binding Pocket ◽  
Trna Synthetase ◽  
Mycobacterium Abscessus ◽  
Drug Binding ◽  
Mycobacterial Species ◽  
In Vitro Susceptibility ◽  
Content Type ◽  
Sequence Variations

ABSTRACT In this study, we aimed to assess the in vitro susceptibility to GSK656 among multiple mycobacterial species and to investigate the correlation between leucyl-tRNA synthetase (LeuRS) sequence variations and in vitro susceptibility to GSK656 among mycobacterial species. A total of 187 mycobacterial isolates, comprising 105 Mycobacterium tuberculosis isolates and 82 nontuberculous mycobacteria (NTM) isolates, were randomly selected for the determination of in vitro susceptibility. For M. tuberculosis, 102 of 105 isolates had MICs of ≤0.5 mg/liter, demonstrating a MIC50 of 0.063 mg/liter and a MIC90 of 0.25 mg/liter. An epidemiological cutoff value of 0.5 mg/liter was proposed for identification of GSK656-resistant M. tuberculosis strains. For NTM, the MIC50 and MIC90 values were >8.0 mg/liter for both Mycobacterium intracellulare and Mycobacterium avium. In contrast, all Mycobacterium abscessus isolates had MICs of ≤0.25 mg/liter, yielding a MIC90 of 0.063 mg/liter. LeuRS from M. abscessus showed greater sequence similarity to M. tuberculosis LeuRS than to LeuRSs from M. avium and M. intracellulare. Sequence alignment revealed 28 residues differing between LeuRSs from M. avium and M. intracellulare and LeuRSs from M. tuberculosis and M. abscessus; among them, 15 residues were in the drug binding domain. Structure modeling revealed that several different residues were close to the tRNA-LeuRS interface or the entrance of the drug-tRNA binding pocket. In conclusion, our data demonstrate significant species diversity in in vitro susceptibility to GSK656 among various mycobacterial species. GSK656 has potent efficacy against M. tuberculosis and M. abscessus, whereas inherent resistance was noted for M. intracellulare and M. avium.

scholarly journals Structural basis for subtype-specific inhibition of the P2X7 receptor

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Akira Karasawa ◽  
Toshimitsu Kawate
Keyword(s):  
P2x7 Receptor ◽  
Hydrophobic Interactions ◽  
Binding Pocket ◽  
Drug Binding ◽  
Structural Basis ◽  
Atp Binding ◽  
Allosteric Site ◽  
Channel Opening ◽  
A Site ◽  
Novel Drug

The P2X7 receptor is a non-selective cation channel activated by extracellular adenosine triphosphate (ATP). Chronic activation of P2X7 underlies many health problems such as pathologic pain, yet we lack effective antagonists due to poorly understood mechanisms of inhibition. Here we present crystal structures of a mammalian P2X7 receptor complexed with five structurally-unrelated antagonists. Unexpectedly, these drugs all bind to an allosteric site distinct from the ATP-binding pocket in a groove formed between two neighboring subunits. This novel drug-binding pocket accommodates a diversity of small molecules mainly through hydrophobic interactions. Functional assays propose that these compounds allosterically prevent narrowing of the drug-binding pocket and the turret-like architecture during channel opening, which is consistent with a site of action distal to the ATP-binding pocket. These novel mechanistic insights will facilitate the development of P2X7-specific drugs for treating human diseases.

scholarly journals Ribosome profiling in Streptococcus pneumoniae reveals the role of methylation of 23S rRNA nucleotide G748 on ribosome stalling

2020 ◽  
Author(s):  
Tatsuma Shoji ◽  
Akiko Takaya ◽  
Yoko Kusuya ◽  
Hiroki Takahashi ◽  
Hiroto Kawashima
Keyword(s):  
Streptococcus Pneumoniae ◽  
Ribosome Profiling ◽  
23S Rrna ◽  
Ribosome Stalling ◽  
70S Ribosome ◽  
Exit Tunnel ◽  
Species Specific ◽  
Peptidyltransferase Center ◽  
First Time

2.Abstract(1) BackgroundMany nucleotides in 23S rRNA are methylated post-transcriptionally by methyltransferases and cluster around the peptidyltransferase center (PTC) and the nascent peptidyl exit tunnel (NPET) located in 50S subunit of 70S ribosome. Biochemical interactions between a nascent peptide and the tunnel may stall ribosome movement and affect expression levels of the protein. However, no studies have shown a role for NPET on ribosome stalling using an NPET mutant.(2) ResultsA ribosome profiling assay in Streptococcus pneumoniae demonstrates for the first time that an NPET mutant exhibits completely different ribosome occupancy compared to wild-type. We demonstrate, using RNA footprinting, that changes in ribosome occupancy correlate with changes in ribosome stalling. Further, statistical analysis shows that short peptide sequences that cause ribosome stalling are species-specific and evolutionarily selected. NPET structure is required to realize these specie-specific ribosome stalling.(3) ConclusionsResults support the role of NPET on ribosome stalling. NPET structure is required to realize the species-specific and evolutionary conserved ribosome stalling. These findings clarify the role of NPET structure on the translation process.

scholarly journals Multidrug Resistance in Neisseria gonorrhoeae: Identification of Functionally Important Residues in the MtrD Efflux Protein

mBio ◽  
2019 ◽  
Vol 10 (6) ◽  
Author(s):  
Mohsen Chitsaz ◽  
Lauren Booth ◽  
Mitchell T. Blyth ◽  
Megan L. O’Mara ◽  
Melissa H. Brown
Keyword(s):  
Molecular Dynamics ◽  
Multidrug Resistance ◽  
Neisseria Gonorrhoeae ◽  
Molecular Dynamics Simulations ◽  
Docking Studies ◽  
Drug Binding ◽  
Multidrug Efflux ◽  
Content Type ◽  
Binding Pockets ◽  
Dynamics Simulations

ABSTRACT A key mechanism that Neisseria gonorrhoeae uses to achieve multidrug resistance is the expulsion of structurally different antimicrobials by the MtrD multidrug efflux protein. MtrD resembles the homologous Escherichia coli AcrB efflux protein with several common structural features, including an open cleft containing putative access and deep binding pockets proposed to interact with substrates. A highly discriminating N. gonorrhoeae strain, with the MtrD and NorM multidrug efflux pumps inactivated, was constructed and used to confirm and extend the substrate profile of MtrD to include 14 new compounds. The structural basis of substrate interactions with MtrD was interrogated by a combination of long-timescale molecular dynamics simulations and docking studies together with site-directed mutagenesis of selected residues. Of the MtrD mutants generated, only one (S611A) retained a wild-type (WT) resistance profile, while others (F136A, F176A, I605A, F610A, F612C, and F623C) showed reduced resistance to different antimicrobial compounds. Docking studies of eight MtrD substrates confirmed that many of the mutated residues play important nonspecific roles in binding to these substrates. Long-timescale molecular dynamics simulations of MtrD with its substrate progesterone showed the spontaneous binding of the substrate to the access pocket of the binding cleft and its subsequent penetration into the deep binding pocket, allowing the permeation pathway for a substrate through this important resistance mechanism to be identified. These findings provide a detailed picture of the interaction of MtrD with substrates that can be used as a basis for rational antibiotic and inhibitor design. IMPORTANCE With over 78 million new infections globally each year, gonorrhea remains a frustratingly common infection. Continuous development and spread of antimicrobial-resistant strains of Neisseria gonorrhoeae, the causative agent of gonorrhea, have posed a serious threat to public health. One of the mechanisms in N. gonorrhoeae involved in resistance to multiple drugs is performed by the MtrD multidrug resistance efflux pump. This study demonstrated that the MtrD pump has a broader substrate specificity than previously proposed and identified a cluster of residues important for drug binding and translocation. Additionally, a permeation pathway for the MtrD substrate progesterone actively moving through the protein was determined, revealing key interactions within the putative MtrD drug binding pockets. Identification of functionally important residues and substrate-protein interactions of the MtrD protein is crucial to develop future strategies for the treatment of multidrug-resistant gonorrhea.

scholarly journals MexY-Promoted Aminoglycoside Resistance in Pseudomonas aeruginosa: Involvement of a Putative Proximal Binding Pocket in Aminoglycoside Recognition

mBio ◽  
2014 ◽  
Vol 5 (2) ◽  
Author(s):  
Calvin Ho-Fung Lau ◽  
Daniel Hughes ◽  
Keith Poole
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Transmembrane Domain ◽  
Binding Pocket ◽  
Drug Binding ◽  
Dimensional Structure ◽  
Efflux System ◽  
Aminoglycoside Resistance ◽  
Content Type ◽  
Export Pathway ◽  
The Impact

ABSTRACTThe resistance-nodulation-division (RND) family multidrug efflux system MexXY-OprM is a major determinant of aminoglycoside resistance inPseudomonas aeruginosa, although the details of aminoglycoside recognition and export by MexY, the substrate-binding RND component of this efflux system, have not been elucidated. To identify regions/residues of MexY important for aminoglycoside resistance, plasmid-bornemexYwas mutagenized and mutations that impaired MexY-promoted aminoglycoside (streptomycin) resistance were identified in a ΔmexYstrain ofP. aeruginosa. Sixty-one streptomycin-sensitivemexYmutants were recovered; among these, 7 unique mutations that yielded wild-type levels of MexY expression were identified. These mutations compromised resistance to additional aminoglycosides and to other antimicrobials and occurred in both the transmembrane and periplasmic regions of the protein. Mapping of the mutated residues onto a 3-dimensional structure of MexY modeled onEscherichia coliAcrB revealed that these tended to occur in regions implicated in general pump operation (transmembrane domain) and MexY trimer assembly (docking domain) and, thus, did not provide insights into aminoglycoside recognition. A region corresponding to a proximal binding pocket connected to a periplasm-linked cleft, part of a drug export pathway of AcrB, was identified in MexY and proposed to play a role in aminoglycoside recognition. To test this, selected residues (K79, D133, and Y613) within this pocket were mutagenized and the impact on aminoglycoside resistance was assessed. Mutations of D133 and Y613 compromised aminoglycoside resistance, while, surprisingly, the K79 mutation enhanced aminoglycoside resistance, confirming a role for this putative proximal binding pocket in aminoglycoside recognition and export.IMPORTANCEBacterial RND pumps do not typically accommodate highly hydrophilic agents such as aminoglycosides, and it is unclear how those, such as MexY, which accommodate these unique substrates, do so. The results presented here indicate that aminoglycosides are likely not captured and exported by this RND pump component in a unique manner but rather utilize a previously defined export pathway that involves a proximal drug-binding pocket that is also implicated in the export of nonaminoglycosides. The observation, too, that a mutation in this pocket enhances MexY-mediated aminoglycoside resistance (K79A), an indication that it is not optimally designed to accommodate these agents, lends further support to earlier proposals that antimicrobials are not the intended pump substrates.

Chemical parameters influencing fine-tuning in the binding of macrolide antibiotics to the ribosomal tunnel

2007 ◽  
Vol 79 (6) ◽  
pp. 955-968 ◽  
Author(s):  
Erez Pyetan ◽  
David Baram ◽  
Tamar Auerbach-Nevo ◽  
Ada Yonath
Keyword(s):  
Deinococcus Radiodurans ◽  
Bacterial Species ◽  
Ribosomal Subunit ◽  
Binding Pocket ◽  
Fine Tuning ◽  
23S Rrna ◽  
Structure Based Drug Design ◽  
Altered Level ◽  
Ribosomal Tunnel ◽  
Binding Pockets

In comparison to existing structural, biochemical, and therapeutical data, the crystal structures of large ribosomal subunit from the eubacterial pathogen model Deinococcus radiodurans in complex with the 14-membered macrolides erythromycylamine, RU69874, and the 16-membered macrolide josamycin, highlighted the similarities and differences in macrolides binding to the ribosomal tunnel. The three compounds occupy the macrolide binding pocket with their desosamine or mycaminose aminosugar, the C4-C7 edge of the macrolactone ring and the cladinose sugar sharing similar positions and orientations, although the latter, known to be unnecessary for antibiotic activity, displays fewer contacts. The macrolactone ring displays altogether few contacts with the ribosome and can, therefore, tilt in order to optimize its interaction with the 23S rRNA. In addition to their contacts with nucleotides of domain V of the 23S RNA, erythromycylamine and RU69874 interact with domain II nucleotide U790, and RU69874 also reaches van der Waals distance from A752, in a fashion similar to that observed for the ketolides telithromycin and cethromycin. The variability in the sequences and consequently the diversity of the conformations of macrolide binding pockets in various bacterial species can explain the drug's altered level of effectiveness on different organisms and is thus an important factor in structure-based drug design.

scholarly journals Mutations within therplDGene of Linezolid-Nonsusceptible Streptococcus pneumoniae Strains Isolated in the United States

2014 ◽  
Vol 58 (4) ◽  
pp. 2459-2462 ◽  
Author(s):  
W. Dong ◽  
S. Chochua ◽  
L. McGee ◽  
D. Jackson ◽  
K. P. Klugman ◽  
...  
Keyword(s):  
United States ◽  
Streptococcus Pneumoniae ◽  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
Structural Changes ◽  
Binding Pocket ◽  
The United States ◽  
Target Site ◽  
23S Rrna ◽  
Content Type

ABSTRACTThree invasiveStreptococcus pneumoniaestrains nonsusceptible to linezolid were isolated in the United States between 2001 and 2012 from the CDC's Active Bacterial Core surveillance. Linezolid binds ribosomal proteins where structural changes within its target site may confer resistance. Our study identified mutations and deletions near the linezolid binding pocket of two of these strains within therplDgene, which encodes ribosomal protein L4. Mutations in the 23S rRNA alleles or therplVgene were not detected.

scholarly journals PyVOL: a PyMOL plugin for visualization, comparison, and volume calculation of drug-binding sites

2019 ◽  
Author(s):  
Ryan H.B. Smith ◽  
Arvin C. Dar ◽  
Avner Schlessinger
Keyword(s):  
Binding Pocket ◽  
Drug Binding ◽  
Identification Algorithm ◽  
Volume Determination ◽  
Volume Calculation ◽  
User Input ◽  
Binding Pockets ◽  
Drug Binding Sites ◽  
Python Package ◽  
Pymol Plugin

AbstractMotivationBinding pocket volumes are a simple yet important predictor of small molecule binding; however, generating visualizations of pocket topology and performing meaningful volume comparisons can be difficult with available tools. Current programs for accurate volume determination rely on extensive user input to define bulk solvent boundaries and to partition cavities into subpockets, increasing inter-user variability in measurements as well as time demands.ResultsWe developed PyVOL, a python package with a PyMOL interface and GUI, to visualize, to characterize, and to compare binding pockets. PyVOL’s pocket identification algorithm is designed to maximize reproducibility through minimization of user-provided parameters, avoidance of grid-based methods, and automated subpocket identification. This approach permits efficient, scalable volume calculations.AvailabilityPyVOL is released under the MIT License. Source code and documentation are available through github (https://github.com/schlessingerlab/pyvol/) with distribution through PyPI (bio-pyvol)[email protected], [email protected]

scholarly journals The SARS-CoV-2 replication-transcription complex is a priority target for broad-spectrum pan-coronavirus drugs

2021 ◽  
Author(s):  
Setayesh Yazdani ◽  
Nicola De Maio ◽  
Yining Ding ◽  
Vijay Shahani ◽  
Nick Goldman ◽  
...  
Keyword(s):  
Broad Spectrum ◽  
Disease Burden ◽  
Rna Binding ◽  
Protein Structures ◽  
Drug Binding ◽  
Binding Pockets ◽  
To Come ◽  
Target Binding ◽  
A Site ◽  
Experimental Structure

ABSTRACTIn the absence of effective treatment, COVID-19 is likely to remain a global disease burden. Compounding this threat is the near certainty that novel coronaviruses with pandemic potential will emerge in years to come. Pan-coronavirus drugs – agents active against both SARS-CoV-2 and other coronaviruses – would address both threats. A strategy to develop such broad-spectrum inhibitors is to pharmacologically target binding sites on SARS-CoV-2 proteins that are highly conserved in other known coronaviruses, the assumption being that any selective pressure to keep a site conserved across past viruses will apply to future ones. Here, we systematically mapped druggable binding pockets on the experimental structure of fifteen SARS-CoV-2 proteins and analyzed their variation across twenty-seven α- and β-coronaviruses and across thousands of SARS-CoV-2 samples from COVID-19 patients. We find that the two most conserved druggable sites are a pocket overlapping the RNA binding site of the helicase nsp13, and the catalytic site of the RNA-dependent RNA polymerase nsp12, both components of the viral replication-transcription complex. We present the data on a public web portal (https://www.thesgc.org/SARSCoV2_pocketome/) where users can interactively navigate individual protein structures and view the genetic variability of drug binding pockets in 3D.

scholarly journals Investigation of Multiple Resistance Mechanisms in Voriconazole-Resistant Aspergillus flavus Clinical Isolates from a Chest Hospital Surveillance in Delhi, India

2018 ◽  
Vol 62 (3) ◽  
Author(s):  
Cheshta Sharma ◽  
Rakesh Kumar ◽  
Nitin Kumar ◽  
Aradhana Masih ◽  
Dinesh Gupta ◽  
...  
Keyword(s):  
Aspergillus Flavus ◽  
Target Genes ◽  
Empirical Therapy ◽  
Homology Model ◽  
Binding Pocket ◽  
Resistance Mechanisms ◽  
Drug Binding ◽  
Whole Genome Sequence ◽  
Azole Resistance ◽  
Content Type

ABSTRACT Invasive and allergic infections by Aspergillus flavus are more common in tropical and subtropical countries. The emergence of voriconazole (VRC) resistance in A. flavus impacts the management of aspergillosis, as azoles are used as the first-line and empirical therapy. We screened 120 molecularly confirmed A. flavus isolates obtained from respiratory and sinonasal specimens in a chest hospital in Delhi, India, for azole resistance using the CLSI broth microdilution (CLSI-BMD) method. Overall, 2.5% ( n = 3/120) of A. flavus isolates had VRC MICs above epidemiological cutoff values (>1 μg/ml). The whole-genome sequence analysis of three non-wild-type (WT) A. flavus isolates with high VRC MICs showed polymorphisms in azole target genes ( cyp51A , cyp51B , and cyp51C ). Further, four novel substitutions (S196F, A324P, N423D, and V465M) encoded in the cyp51C gene were found in a single non-WT isolate which also exhibited overexpression of cyp51 ( cyp51A , - B , and - C ) genes and transporter genes, namely, MDR1 , MDR2 , atrF , and mfs1 . The homology model of the non-WT isolate suggests that substitutions S196F and N423D exhibited major structural and functional effects on cyp51C drug binding. The substrate (drug) may not be able to bind to binding pocket due to changes in the pocket size or closing down or narrowing of cavities in drug entry channels. Notably, the remaining two VRC-resistant A. flavus isolates, including the one which had a pan-azole resistance phenotype (itraconazole and posaconazole), did not show upregulation of any of the analyzed target genes. These results suggest that multiple target genes and mechanisms could simultaneously contribute to azole resistance in A. flavus .

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