{"doi":"10.1016/j.jbc.2021.101188","title":"Mutations in PBP2 from ceftriaxone-resistant Neisseria gonorrhoeae alter the dynamics of the β3–β4 loop to favor a low-affinity drug-binding state","abstract":"Resistance to the extended-spectrum cephalosporin ceftriaxone in the pathogenic bacteria Neisseria gonorrhoeae is conferred by mutations in penicillin-binding protein 2 (PBP2), the lethal target of the antibiotic, but how these mutations exert their effect at the molecular level is unclear. Using solution NMR, X-ray crystallography, and isothermal titration calorimetry, we report that WT PBP2 exchanges dynamically between a low-affinity state with an extended β3–β4 loop conformation and a high-affinity state with an inward β3–β4 loop conformation. Histidine-514, which is located at the boundary of the β4 strand, plays an important role during the exchange between these two conformational states. We also find that mutations present in PBP2 from H041, a ceftriaxone-resistant strain of N. gonorrhoeae, increase resistance to ceftriaxone by destabilizing the inward β3–β4 loop conformation or stabilizing the extended β3–β4 loop conformation to favor the low-affinity drug-binding state. These observations reveal a unique mechanism for ceftriaxone resistance, whereby mutations in PBP2 lower the proportion of target molecules in the high-affinity drug-binding state and thus reduce inhibition at lower drug concentrations. Resistance to the extended-spectrum cephalosporin ceftriaxone in the pathogenic bacteria Neisseria gonorrhoeae is conferred by mutations in penicillin-binding protein 2 (PBP2), the lethal target of the antibiotic, but how these mutations exert their effect at the molecular level is unclear. Using solution NMR, X-ray crystallography, and isothermal titration calorimetry, we report that WT PBP2 exchanges dynamically between a low-affinity state with an extended β3–β4 loop conformation and a high-affinity state with an inward β3–β4 loop conformation. Histidine-514, which is located at the boundary of the β4 strand, plays an important role during the exchange between these two conformational states. We also find that mutations present in PBP2 from H041, a ceftriaxone-resistant strain of N. gonorrhoeae, increase resistance to ceftriaxone by destabilizing the inward β3–β4 loop conformation or stabilizing the extended β3–β4 loop conformation to favor the low-affinity drug-binding state. These observations reveal a unique mechanism for ceftriaxone resistance, whereby mutations in PBP2 lower the proportion of target molecules in the high-affinity drug-binding state and thus reduce inhibition at lower drug concentrations. Neisseria gonorrhoeae is the causative agent of the sexually transmitted infection gonorrhea, with nearly 80 million cases worldwide each year (1Newman L. Rowley J. Vander Hoorn S. Wijesooriya N.S. Unemo M. Low N. Stevens G. Gottlieb S. Kiarie J. Temmerman M. Global estimates of the prevalence and incidence of four curable sexually transmitted infections in 2012 based on systematic review and global reporting.PLoS One. 2015; 10e0143304Crossref PubMed Scopus (908) Google Scholar). Without antibiotic treatment, infections persist as a chronic disease and can cause serious sequelae, including pelvic inflammatory disease, infertility, arthritis, and disseminated infections (2Hill S.A. Masters T.L. Wachter J. Gonorrhea - an evolving disease of the new millennium.Microb. Cell. 2016; 3: 371-389Crossref PubMed Scopus (41) Google Scholar). For many years, N. gonorrhoeae was treated with a single dose of penicillin, and more recently, ceftriaxone. In 2012, the emergence of several high-level ceftriaxone-resistant strains led the Centers for Disease Control and Prevention to change its recommended treatment for gonorrhea from monotherapy to dual therapy with ceftriaxone and azithromycin (3Workowski K.A. Bolan G.A. Centers for Disease Control and PreventionSexually transmitted diseases treatment guidelines, 2015.MMWR Recomm. Rep. 2015; 64: 1-137Crossref PubMed Scopus (25) Google Scholar, 4Unemo M. Lahra M.M. Cole M. Galarza P. Ndowa F. Martin I. Dillon J.R. Ramon-Pardo P. Bolan G. Wi T. World Health Organization Global Gon","journal":"Journal of Biological Chemistry","year":2021,"id":174775,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":23,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9521,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2021-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":340805,"name":"Joshua Tomberg","orcid":null,"position":1,"is_corresponding":false},{"id":714929,"name":"Carly A. Sciandra","orcid":null,"position":2,"is_corresponding":false},{"id":339442,"name":"Robert A. Nicholas","orcid":"0000-0003-2507-8974","position":3,"is_corresponding":false},{"id":339443,"name":"Christopher Davies","orcid":"0000-0002-6687-4064","position":4,"is_corresponding":false},{"id":494202,"name":"Pei Zhou","orcid":"0000-0002-7823-3416","position":5,"is_corresponding":false},{"id":494201,"name":"Benjamin Fenton","orcid":"0000-0001-8583-8256","position":0,"is_corresponding":true}],"reference_count":41,"raw_metadata":null,"created_at":"2026-07-18T23:47:06.584146Z","pmid":"34529975","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}