Abstract
<jats:p>Mosaic penA alleles encoding highly mutated variants of penicillin-binding protein 2 (PBP2) are the principal determinants of ceftriaxone resistance in Neisseria gonorrhoeae. Resistance-associated mutations in PBP2 from the ceftriaxone-resistant strain H041 restrict formation of the inward conformation of the β3-β4 loop associated with efficient acylation, but how β-lactam recognition is coupled to this conformational switch is unknown. Because the conserved active-site residue Tyr422 interacts with the R1 substituent of β-lactams, we investigated its role in coupling ligand recognition and acylation activity. Mutation of Tyr422 to Ala lowered acylation rates by up to 120-fold for cefoperazone and piperacillin, whereas acylation rates of ceftriaxone increased 4-fold. Unexpectedly, the crystal structure of the Y422A mutant acylated by ceftriaxone revealed that the β3-β4 loop had adopted the inward, high-activity conformation, despite position 422 being spatially distant from the loop. Transformation experiments showed that cell viability requires a tyrosine at position 422, indicating the residue is essential for transpeptidase function. Together, these findings reveal an energetic coupling between an active-site residue in PBP2 and a conformational switch whose equilibrium is altered by resistance mutations. The previously observed higher activity of β-lactams containing extended R1 groups is consistent with stronger interactions with Tyr422 that favor the conformational switch. Molecular modeling suggests that such groups enhance activity by mimicking the iso-Glu region of the pentapeptide substrate. Overall, we propose that access to the high-activity state of PBP2 where the β3-β4 loop is inward is regulated by interactions between Tyr422 and β-lactam R1 groups, and that resistance mutations function by tilting the balance toward a lower activity state.</jats:p>