{"doi":"10.1016/j.jbc.2025.110850","title":"A β-lactamase inhibitory protein mutant displays high potency and a broad inhibition profile due to an altered binding mode with β-lactamases","abstract":"β-lactamase enzymes inactivate β-lactam antibiotics, leading to drug resistance. The β-lactamase inhibitory protein (BLIP) is a naturally occurring inhibitor of β-lactamases, with inhibition constants ( K i ) ranging from picomolar to micromolar values. For example, BLIP inhibits CTX-M-14 β-lactamase with a K i of 330 nM while the K i for CTX-M-15 is 3 nM, despite CTX-M-14 and CTX-M-15 sharing 83% sequence identity. We used a genetic screen to identify a BLIP mutant, E73W, that potently inhibited CTX-M-14. Subsequent purification and testing of BLIP E73W revealed it is a potent, broad-spectrum inhibitor of class A β-lactamases. We determined structures of BLIP E73W in complex with the CTX-M-14, CTX-M-15, and TEM-1 β-lactamases to investigate the basis of the broad-spectrum inhibition. Previous structures of BLIP in complex with several class-A β-lactamases revealed that β-lactamase active site residue Tyr105 is found in an altered rotamer conformation. Also, in the case of the BLIP/CTX-M-15 complex, an altered conformation of the active site 103-106 loop is observed. In contrast, the BLIP E73W/β-lactamase complexes did not show the altered conformations of Tyr105 or the 103-106 loop. Instead, the mutant's mechanism involves BLIP Trp73 trapping Tyr105 against the wall of the active site in a similar conformation as in the apo-enzyme. Interestingly, the E73W mutant binds the apo-enzyme conformation in all of the BLIP E73W/β-lactamase complexes. Binding to the apo-enzyme conformation, which is expected to be highly populated in solution, as well as enhanced hydrophobic interactions of Trp73 with β-lactamases are possible explanations for the high potency and broad-spectrum inhibition.","journal":"Journal of Biological Chemistry","year":2025,"id":578846,"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":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9535,"is_data_producer":true,"deposit_databanks":{"PDB":["9Q0C","9Q0B","9Q0A"]},"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":399418,"name":"Shuo Lu","orcid":"0000-0002-5573-5501","position":1,"is_corresponding":false},{"id":1489114,"name":"Dignite Ngango","orcid":null,"position":2,"is_corresponding":false},{"id":249510,"name":"Banumathi Sankaran","orcid":"0000-0002-3266-8131","position":3,"is_corresponding":false},{"id":399420,"name":"B. V. Venkataram Prasad","orcid":"0000-0002-1172-2071","position":4,"is_corresponding":false},{"id":338702,"name":"Timothy Palzkill","orcid":"0000-0002-5267-0001","position":5,"is_corresponding":false},{"id":902102,"name":"Paola Rivera","orcid":"0000-0002-6437-5210","position":0,"is_corresponding":true}],"reference_count":52,"raw_metadata":null,"created_at":"2026-07-19T02:58:24.957414Z","pmid":"41135675","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":[]}