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Publication Abstract from PubMed

beta-lactamase enzymes inactivate beta-lactam antibiotics, leading to drug resistance. The beta-lactamase inhibitory protein (BLIP) is a naturally occurring inhibitor of beta-lactamases, with inhibition constants (K(i)) ranging from picomolar to micromolar values. For example, BLIP inhibits CTX-M-14 beta-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 beta-lactamases. We determined structures of BLIP E73W in complex with the CTX-M-14, CTX-M-15, and TEM-1 beta-lactamases to investigate the basis of the broad-spectrum inhibition. Previous structures of BLIP in complex with several class-A beta-lactamases revealed that beta-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/beta-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/beta-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 beta-lactamases are possible explanations for the high potency and broad-spectrum inhibition.

A beta-lactamase inhibitory protein mutant displays high potency and a broad inhibition profile due to an altered binding mode with beta-lactamases.,Rivera P, Lu S, Ngango D, Sankaran B, Venkataram Prasad BV, Palzkill T J Biol Chem. 2025 Oct 22:110850. doi: 10.1016/j.jbc.2025.110850. PMID:41135675^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.