A novel inhibitor of the new antibiotic resistance protein OptrA

Zhong, Xiaobo; Xiang, Hua; Wang, Tiedong; Zhong, Ling; Ming, Di; Nie, Linyan; Cao, Fengjiao; Li, Bangbang; Cao, Junsheng; Mu, Dan; Ruan, Ke; Wang, Lin; Wang, Dacheng

doi:10.1111/cbdd.13311

Cited by 11 publications

(6 citation statements)

References 26 publications

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“…Such EQ2 variants have been successfully employed to trap other ABCF proteins on the ribosome because they allow binding but prevent hydrolysis of ATP (Chen et al, 2014;Crowe-McAuliffe et al, 2018;Crowe-McAuliffe et al, 2021;Kasari et al, 2019). The OptrA-EQ2 variant was earlier shown to have a compromised in vitro ATPase activity and be incompetent in promoting antibotic resistance in vivo (Zhong et al, 2018). Affinity purification via the FLAG3 tag was performed in the presence of 0.5 mM ATP from clarified lysates prepared from the E. faecalis DlsaA strain expressing the FLAG3-tagged ARE-ABCF, either wildtype or EQ2-variant.…”

Section: Cryo-em Structures Of Poxta-70s Complexesmentioning

confidence: 99%

Structural basis for PoxtA-mediated resistance to Phenicol and Oxazolidinone antibiotics

Crowe‐McAuliffe

Murina

Kasari

et al. 2021

Preprint

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PoxtA and OptrA are ATP binding cassette (ABC) proteins of the F subtype (ABCF) that confer resistance to oxazolidinone, such as linezolid, and phenicol antibiotics, such as chloramphenicol. PoxtA/OptrA are often encoded on mobile genetic elements, facilitating their rapid spread amongst Gram-positive bacteria. These target protection proteins are thought to confer resistance by binding to the ribosome and dislodging the antibiotics from their binding sites. However, a structural basis for their mechanism of action has been lacking. Here we present cryo-electron microscopy structures of PoxtA in complex with the Enterococcus faecalis 70S ribosome at 2.9-3.1A, as well as the complete E. faecalis 70S ribosome at 2.2-2.5 A. The structures reveal that PoxtA binds within the ribosomal E-site with its antibiotic resistance domain (ARD) extending towards the peptidyltransferase center (PTC) on the large ribosomal subunit. At its closest point, the ARD of PoxtA is still located >15A from the linezolid and chloramphenicol binding sites, suggesting that drug release is elicited indirectly. Instead, we observe that the ARD of PoxtA perturbs the CCA-end of the P-site tRNA causing it to shift by ~4A out of the PTC, which correlates with a register shift of one amino acid for the attached nascent polypeptide chain. Given that linezolid and chloramphenicol are context-specific translation elongation inhibitors, we postulate that PoxtA/OptrA confer resistance to oxazolidinones and phenicols indirectly by perturbing the P-site tRNA and thereby altering the conformation of the attached nascent chain to disrupt the drug binding site.

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Section: Cryo-em Structures Of Poxta-70s Complexesmentioning

confidence: 99%

Structural basis for PoxtA-mediated resistance to Phenicol and Oxazolidinone antibiotics

Crowe‐McAuliffe

Murina

Kasari

et al. 2021

Preprint

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“…Nevertheless, a possible approach could be small fragment library screening followed by elucidation of ligand‐bound ARE ABC‐F structure and modification of ligand to outcompete the natural substrate, such as ATP, for disabling the catalytically important conserved signature motif and Walker B following glutamate residues. Recently, a novel inhibitor of OptrA that targets its ATPase center was discovered . Through saturation transfer difference experiments, homology modeling of OptrA, docking, and molecular dynamics, the authors have proposed the binding site interactions of their inhibitor lead for structural optimization beyond its reported 30% ATPase activity suppression.…”

Section: Implications For Drug Designmentioning

confidence: 99%

Ribosome protection by ABC‐F proteins—Molecular mechanism and potential drug design

Ero

Kumar

et al. 2019

Protein Science

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Members of the ATP-binding cassette F (ABC-F) proteins confer resistance to several classes of clinically important antibiotics through ribosome protection. Recent structures of two ABC-F proteins, Pseudomonas aeruginosa MsrE and Bacillus subtilis VmlR bound to ribosome have shed light onto the ribosome protection mechanism whereby drug resistance is mediated by the antibiotic resistance domain (ARD) connecting the two ATP binding domains. ARD of the E site bound MsrE and VmlR extends toward the drug binding region within the peptidyl transferase center (PTC) and leads to conformational changes in the P site tRNA acceptor stem, the PTC, and the drug binding site causing the release of corresponding drugs. The structural similarities and differences of the MsrE and VmlR structures likely highlight an universal ribosome protection mechanism employed by antibiotic resistance (ARE) ABC-F proteins. The variable ARD domains enable this family of proteins to adapt the protection mechanism for several classes of ribosome-targeting drugs. ARE ABC-F genes have been found in numerous pathogen genomes and multi-drug resistance conferring plasmids. Collectively they mediate resistance to a broader range of antimicrobial agents than any other group of resistance proteins and play a major role in clinically significant drug resistance in pathogenic bacteria. Here, we review the recent structural and biochemical findings on these emerging resistance proteins, offering an update of the molecular basis and implications for overcoming ABC-F conferred drug resistance.

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“…10,11 The optrA, found in 2015, was reported for its ATP hydrolysis activity to be associated with linezolid resistance. 12 Also, previous study reported that there were different amino acid substitutions in optrA. 13 In this study, we investigated the molecular characterization of optrApositive enterococci and explored the function of varied types of optrA on the linezolid susceptibility.…”

Section: Introductionmentioning

confidence: 99%

<p>Molecular Investigations of Linezolid Resistance in Enterococci <em>OptrA</em> Variants from a Hospital in Shanghai</p>

Yang

et al. 2020

IDR

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Purpose: The OptrA protein is a member of ATP-binding cassette (ABC) transporters, a transporter family which can confer resistance to oxazolidinone antibiotics by transferring plasmid. We aim to describe the distribution of optrA-harbored Enterococcus in Huashan hospital in 2017 and to address the effects of optrA mutations on the susceptibility of linezolid antibiotic drug. Methods: Linezolid-resistance-related genes were tested for Enterococcus by polymerase chain reaction (PCR) and then sequenced for amino acid substitution site analysis. Broth microdilution and agar dilution test were applied to determine the minimal inhibitory concentration (MIC) of linezolid for Enterococcus containing optrA. Pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were used to evaluate the genotypes of optrA-positive isolates. To verify the functions of two main optrA variants, optrA over-expressing strains were constructed. Results: Among 20 optrA-positive strains, only two were resistant to linezolid. No amino acid substitution existed in 23S rRNA V domain among Enterococcus faecalis. None had cfr, cfr(B) or cfr(C) genes. F101L and G4D/K/R or T150A were the main substitutions of ribosomal protein L4, L3, respectively. We found one Enterococcus faecium isolate cocontained optrA and poxtA and another E. faecalis isolate co-contained optrA and cfr(D), but they were not resistant to linezolid. Among 20 optrA-positive strains, ST-16 was the main type. Two main optrA variants KD (T112K, Y176D) and RDK (I104R, Y176D, E256K) slightly raised enterococci's MIC of linezolid. Conclusion: OptrA exists in linezolid non-resistant enterococci with diverse amino acid substitutions. The variants play different roles in changing the MIC of linezolid.

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A novel inhibitor of the new antibiotic resistance protein OptrA

Cited by 11 publications

References 26 publications

Structural basis for PoxtA-mediated resistance to Phenicol and Oxazolidinone antibiotics

Structural basis for PoxtA-mediated resistance to Phenicol and Oxazolidinone antibiotics

Ribosome protection by ABC‐F proteins—Molecular mechanism and potential drug design

<p>Molecular Investigations of Linezolid Resistance in Enterococci <em>OptrA</em> Variants from a Hospital in Shanghai</p>

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