{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,8,12]],"date-time":"2026-08-12T05:28:51Z","timestamp":1786512531523,"version":"build-2736575974"},"posted":{"date-parts":[[2026,8,10]]},"group-title":"Biochemistry","reference-count":38,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,8,10]],"date-time":"2026-08-10T00:00:00Z","timestamp":1786320000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"funder":[{"award":["AI164794"],"award-info":[{"award-number":["AI164794"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["AI113170"],"award-info":[{"award-number":["AI113170"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["AI153521"],"award-info":[{"award-number":["AI153521"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,8,10]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Mosaic\n                  <jats:italic>penA<\/jats:italic>\n                  alleles encoding highly mutated variants of penicillin-binding protein 2 (PBP2) are the principal determinants of ceftriaxone resistance in\n                  <jats:italic>Neisseria gonorrhoeae<\/jats:italic>\n                  . Resistance-associated mutations in PBP2 from the ceftriaxone-resistant strain H041 restrict formation of the inward conformation of the \u03b23\u2013\u03b24 loop associated with efficient acylation, but how \u03b2-lactam recognition is coupled to this conformational switch is unknown. Because the conserved active-site residue Tyr422 interacts with the R\n                  <jats:sub>1<\/jats:sub>\n                  substituent of \u03b2-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 \u03b23\u2013\u03b24 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 \u03b2-lactams containing extended R\n                  <jats:sub>1<\/jats:sub>\n                  groups is consistent with stronger interactions with Tyr422 that favor the conformational switch. Molecular modeling suggests that such groups enhance activity by mimicking the\n                  <jats:italic>iso<\/jats:italic>\n                  -Glu region of the pentapeptide substrate. Overall, we propose that access to the high-activity state of PBP2 where the \u03b23\u2013\u03b24 loop is inward is regulated by interactions between Tyr422 and \u03b2-lactam R\n                  <jats:sub>1<\/jats:sub>\n                  groups, and that resistance mutations function by tilting the balance toward a lower activity state.\n                <\/jats:p>","DOI":"10.64898\/2026.08.07.743578","type":"posted-content","created":{"date-parts":[[2026,8,10]],"date-time":"2026-08-10T12:25:16Z","timestamp":1786364716000},"source":"Crossref","is-referenced-by-count":0,"title":["Energetic coupling of an active site residue in penicillin-binding protein 2 from\n                  <i>Neisseria gonorrhoeae<\/i>\n                  with a resistance-associated conformational switch in the \u03b23-\u03b24 loop"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7955-795X","authenticated-orcid":false,"given":"Caleb M.","family":"Stratton","sequence":"first","affiliation":[{"name":"University of South Alabama, Department of Biochemistry and Molecular Biology, Mobile, Alabama, 36688, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sandeepchowdary","family":"Bala","sequence":"additional","affiliation":[{"name":"University of South Alabama, Department of Biochemistry and Molecular Biology, Mobile, Alabama, 36688, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marissa M.","family":"Bivins","sequence":"additional","affiliation":[{"name":"University of North Carolina at Chapel Hill, Department of Pharmacology, Chapel Hill, North Carolina, 27599, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Robert A.","family":"Nicholas","sequence":"additional","affiliation":[{"name":"University of North Carolina at Chapel Hill, Department of Pharmacology, Chapel Hill, North Carolina, 27599, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christopher","family":"Davies","sequence":"additional","affiliation":[{"name":"University of South Alabama, Department of Biochemistry and Molecular Biology, Mobile, Alabama, 36688, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026081121501267000_2026.08.07.743578v1.1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.RA119.009942"},{"key":"2026081121501267000_2026.08.07.743578v1.2","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.RA120.012617"},{"key":"2026081121501267000_2026.08.07.743578v1.3","unstructured":"Centers for Disease Control & Prevention. 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