{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,2]],"date-time":"2026-09-02T08:14:52Z","timestamp":1788336892558,"version":"build-2803163510"},"reference-count":33,"publisher":"American Chemical Society (ACS)","issue":"48","license":[{"start":{"date-parts":[[2015,11,18]],"date-time":"2015-11-18T00:00:00Z","timestamp":1447804800000},"content-version":"vor","delay-in-days":365,"URL":"https:\/\/pubs.acs.org\/page\/policy\/authorchoice_termsofuse.html"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["AI36901"],"award-info":[{"award-number":["AI36901"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["GM66861"],"award-info":[{"award-number":["GM66861"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2014,12,9]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>A hallmark of penicillin-binding protein 2 (PBP2) from penicillin-resistant strains of Neisseria gonorrhoeae is insertion of an aspartate after position 345. The insertion resides on a loop near the active site and is immediately adjacent to an existing aspartate (Asp346) that forms a functionally important hydrogen bond with Ser363 of the SxN conserved motif. Insertion of other amino acids, including Glu and Asn, can also lower the rate of acylation by penicillin, but these insertions abolish transpeptidase function. Although the kinetic consequences of the Asp insertion are well-established, how it impacts the structure of PBP2 is unknown. Here, we report the 2.2 \u00c5 resolution crystal structure of a truncated construct of PBP2 containing all five mutations present in PBP2 from the penicillin-resistant strain 6140, including the Asp insertion. Commensurate with the strict specificity for the Asp insertion over similar amino acids, the insertion does not cause disordering of the structure, but rather induces localized flexibility in the \u03b22c\u2212\u03b22d loop. The crystal structure resolves the ambiguity of whether the insertion is Asp345a or Asp346a (due to the adjacent Asp) because the hydrogen bond between Asp346 and Ser362 is preserved and the insertion is therefore Asp346a. The side chain of Asp346a projects directly toward the \u03b2-lactam-binding site near Asn364 of the SxN motif. The Asp insertion may lower the rate of acylation by sterically impeding binding of the antibiotic or by hindering breakage of the \u03b2-lactam ring during acylation because of the negative charge of its side chain.<\/jats:p>","DOI":"10.1021\/bi5011317","type":"journal-article","created":{"date-parts":[[2014,11,18]],"date-time":"2014-11-18T07:14:55Z","timestamp":1416294895000},"page":"7596-7603","source":"Crossref","is-referenced-by-count":25,"title":["Structural Effect of the Asp345a Insertion in Penicillin-Binding\nProtein 2 from Penicillin-Resistant Strains of\n                    <i>Neisseria gonorrhoeae<\/i>"],"prefix":"10.1021","volume":"53","author":[{"given":"Alena","family":"Fedarovich","sequence":"first","affiliation":[{"name":"Medical University of South Carolina , , , ,","place":["Charleston, South Carolina, United States, 29425"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Edward","family":"Cook","sequence":"additional","affiliation":[{"name":"Medical University of South Carolina , , , ,","place":["Charleston, South Carolina, United States, 29425"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Joshua","family":"Tomberg","sequence":"additional","affiliation":[{"name":"University of North Carolina at Chapel Hill , , , ,","place":["Chapel Hill, North Carolina, United States, 27599"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Robert A.","family":"Nicholas","sequence":"additional","affiliation":[{"name":"University of North Carolina at Chapel Hill , , , ,","place":["Chapel Hill, North Carolina, United States, 27599"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christopher","family":"Davies","sequence":"additional","affiliation":[{"name":"Medical University of South Carolina , , , ,","place":["Charleston, South Carolina, United States, 29425"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2014,11,18]]},"reference":[{"key":"2026090203264810000_cit1","first-page":"590","article-title":"Update to CDC\u2019s Sexually Transmitted Diseases\nTreatment Guidelines, 2010: Oral Cephalosporins No Longer a Recommended\nTreatment for Gonococcal Infections","volume":"61","author":"The Centers for Disease Control and Prevention.","year":"2012","journal-title":"Morbidity\nand Mortality Weekly Report"},{"key":"2026090203264810000_cit2","doi-asserted-by":"crossref","first-page":"2528","DOI":"10.1128\/AAC.02608-13","article-title":"Azithromycin resistance\nis co-evolving with reduced susceptibility to cephalosporins in Neisseria gonorrhoeae, Ontario, Canada","volume":"58","author":"Allen","year":"2014","journal-title":"Antimicrob. Agents Chemother."},{"key":"2026090203264810000_cit3","doi-asserted-by":"crossref","first-page":"148","DOI":"10.3201\/eid1701.100397","article-title":"Ceftriaxone-resistant Neisseria gonorrhoeae, Japan","volume":"17","author":"Ohnishi","year":"2011","journal-title":"Emerging Infect.\nDis."},{"key":"2026090203264810000_cit4","doi-asserted-by":"crossref","first-page":"3538","DOI":"10.1128\/AAC.00325-11","article-title":"Is Neisseria gonorrhoeae initiating a future era of untreatable\ngonorrhea?: Detailed characterization of the first strain with high-level\nresistance to ceftriaxone","volume":"55","author":"Ohnishi","year":"2011","journal-title":"Antimicrob. Agents\nChemother."},{"key":"2026090203264810000_cit5","doi-asserted-by":"crossref","first-page":"1273","DOI":"10.1128\/AAC.05760-11","article-title":"High-level\ncefixime- and ceftriaxone-resistant Neisseria gonorrhoeae in France: Novel penA mosaic allele\nin a successful international clone causes treatment failure","volume":"56","author":"Unemo","year":"2012","journal-title":"Antimicrob. Agents Chemother."},{"key":"2026090203264810000_cit6","doi-asserted-by":"crossref","first-page":"673","DOI":"10.1111\/j.1574-6976.2006.00024.x","article-title":"Penicillin\nbinding proteins: Key players in bacterial cell cycle and drug resistance\nprocesses","volume":"30","author":"Macheboeuf","year":"2006","journal-title":"FEMS Microbiol. Rev."},{"key":"2026090203264810000_cit7","doi-asserted-by":"crossref","first-page":"234","DOI":"10.1111\/j.1574-6976.2008.00105.x","article-title":"The penicillin-binding\nproteins: Structure and role in peptidoglycan biosynthesis","volume":"32","author":"Sauvage","year":"2008","journal-title":"FEMS Microbiol. Rev."},{"key":"2026090203264810000_cit8","doi-asserted-by":"crossref","first-page":"397","DOI":"10.1007\/978-1-4614-1400-1_11","article-title":"Structural Mechanisms of \u03b2-Lactam Antibiotic Resistance in\nPenicillin-Binding Proteins","volume-title":"Antibiotic\nDiscovery & Development","author":"Nicholas","year":"2012"},{"key":"2026090203264810000_cit9","doi-asserted-by":"crossref","first-page":"316","DOI":"10.1128\/AAC.19.2.316","article-title":"Properties\nof penicillin-binding proteins in Neisseria gonorrhoeae","volume":"19","author":"Barbour","year":"1981","journal-title":"Antimicrob. 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Microbiol."},{"key":"2026090203264810000_cit13","first-page":"245","article-title":"Molecular mechanisms of antibiotic\nresistance expressed by the pathogenic\nNeisseriae","volume-title":"Neisseria: Molecular Mechanisms\nof Pathogenesis","author":"Shafer","year":"2010"},{"key":"2026090203264810000_cit14","doi-asserted-by":"crossref","first-page":"173","DOI":"10.1038\/332173a0","article-title":"Hybrid\npenicillin-binding proteins in penicillin-resistant strains of Neisseria gonorrhoeae","volume":"332","author":"Spratt","year":"1988","journal-title":"Nature"},{"key":"2026090203264810000_cit15","doi-asserted-by":"crossref","first-page":"35","DOI":"10.1111\/j.1365-2958.1989.tb00101.x","article-title":"Penicillin-binding\nprotein 2 genes of non-\u03b2-lactamase-producing, penicillin-resistant\nstrains of Neisseria gonorrhoeae","volume":"3","author":"Dowson","year":"1989","journal-title":"Mol. Microbiol."},{"key":"2026090203264810000_cit16","doi-asserted-by":"crossref","first-page":"913","DOI":"10.1111\/j.1365-2958.1990.tb00664.x","article-title":"Insertion\nof an extra amino acid is the main cause of the low affinity of penicillin-binding\nprotein 2 in penicillin-resistant strains of Neisseria gonorrhoeae","volume":"4","author":"Brannigan","year":"1990","journal-title":"Mol. Microbiol."},{"key":"2026090203264810000_cit17","doi-asserted-by":"crossref","first-page":"1202","DOI":"10.1074\/jbc.M805761200","article-title":"Crystal structures\nof penicillin-binding protein 2 from penicillin-susceptible and -resistant\nstrains of Neisseria gonorrhoeae reveal an unexpectedly\nsubtle mechanism for antibiotic resistance","volume":"284","author":"Powell","year":"2009","journal-title":"J. Biol. Chem."},{"key":"2026090203264810000_cit18","doi-asserted-by":"crossref","first-page":"2775","DOI":"10.1021\/bi2017987","article-title":"A highly\nconserved interaction involving the middle residue of the SXN active-site\nmotif is crucial for function of class B penicillin-binding proteins:\nMutational and computational analysis of PBP 2 from N. gonorrhoeae","volume":"51","author":"Tomberg","year":"2012","journal-title":"Biochemistry"},{"key":"2026090203264810000_cit19","doi-asserted-by":"crossref","first-page":"856","DOI":"10.1128\/AAC.30.6.856","article-title":"Genetics of resistance in a non-\u03b2-lactamase-producing\ngonococcus with relatively high-level penicillin resistance","volume":"30","author":"Faruki","year":"1986","journal-title":"Antimicrob. Agents Chemother."},{"key":"2026090203264810000_cit20","doi-asserted-by":"crossref","first-page":"148","DOI":"10.1007\/978-94-011-2928-2_5","article-title":"Mode of action: Interaction with the penicillin-binding\nproteins","volume-title":"The Chemistry of \u03b2-Lactams","author":"Frere","year":"1992"},{"key":"2026090203264810000_cit21","doi-asserted-by":"crossref","first-page":"307","DOI":"10.1016\/S0076-6879(97)76066-X","article-title":"Processing of X-ray\ndiffraction data collected in oscillation mode","volume":"276","author":"Otwinowski","year":"1997","journal-title":"Methods Enzymol."},{"key":"2026090203264810000_cit22","doi-asserted-by":"crossref","first-page":"658","DOI":"10.1107\/S0021889807021206","article-title":"Phaser crystallographic software","volume":"40","author":"McCoy","year":"2007","journal-title":"J. Appl. Crystallogr."},{"key":"2026090203264810000_cit23","first-page":"240","article-title":"Refinement of macromolecular structures\nby the maximum-likelihood method","volume":"D53","author":"Murshudov","year":"1997","journal-title":"Acta Crystallogr."},{"key":"2026090203264810000_cit24","doi-asserted-by":"crossref","first-page":"110","DOI":"10.1107\/S0108767390010224","article-title":"Improved methods\nfor building protein structures in electron-density maps and the location\nof errors in these models","volume":"A47","author":"Jones","year":"1991","journal-title":"Acta Crystallogr."},{"key":"2026090203264810000_cit25","doi-asserted-by":"crossref","first-page":"283","DOI":"10.1107\/S0021889892009944","article-title":"PROCHECK: A program\nto check the stereochemical quality of protein structures","volume":"26","author":"Laskowski","year":"1993","journal-title":"J. Appl. Crystallogr."},{"key":"2026090203264810000_cit26","doi-asserted-by":"crossref","first-page":"616","DOI":"10.1074\/jbc.M004471200","article-title":"Crystal structure of a deacylation-defective\nmutant of penicillin-binding protein 5 at 2.3 \u00c5 resolution","volume":"276","author":"Davies","year":"2001","journal-title":"J. Biol. Chem."},{"key":"2026090203264810000_cit27","doi-asserted-by":"crossref","first-page":"357","DOI":"10.1042\/bj3030357","article-title":"Site-directed\nmutagenesis of proposed active-site residues of penicillin-binding\nprotein 5 from Escherichia coli","volume":"303","author":"van der Linden","year":"1994","journal-title":"Biochem. J."},{"key":"2026090203264810000_cit28","doi-asserted-by":"crossref","first-page":"292","DOI":"10.1016\/S0167-4838(02)00311-4","article-title":"pH, inhibitor, and\nsubstrate specificity studies on Escherichia coli penicillin-binding protein 5","volume":"1597","author":"Stefanova","year":"2002","journal-title":"Biochim. Biophys.\nActa"},{"key":"2026090203264810000_cit29","doi-asserted-by":"crossref","first-page":"52826","DOI":"10.1074\/jbc.M310177200","article-title":"Crystal structure\nof wild-type penicillin-binding protein 5 from E. coli: Implications for deacylation of the acyl-enzyme complex","volume":"278","author":"Nicholas","year":"2003","journal-title":"J. Biol. Chem."},{"key":"2026090203264810000_cit30","doi-asserted-by":"crossref","first-page":"1199","DOI":"10.1021\/bi0268955","article-title":"The crystal\nstructure of phosphonate-inhibited d-Ala-d-Ala peptidase\nreveals an analogue of a tetrahedral transition state","volume":"42","author":"Silvaggi","year":"2003","journal-title":"Biochemistry"},{"key":"2026090203264810000_cit31","doi-asserted-by":"crossref","first-page":"946","DOI":"10.1107\/S0021889891004399","article-title":"MOLSCRIPT:\nA program to produce both detailed and schematic plots of protein\nstructures","volume":"24","author":"Kraulis","year":"1991","journal-title":"J. Appl. Crystallogr."},{"key":"2026090203264810000_cit32","first-page":"869","article-title":"Raster3D version\n2.0. A program for photorealistic\nmolecular graphics","volume":"D50","author":"Merritt","year":"1994","journal-title":"Acta Crystallogr."},{"key":"2026090203264810000_cit33","doi-asserted-by":"crossref","first-page":"1605","DOI":"10.1002\/jcc.20084","article-title":"UCSF Chimera:\nA visualization system\nfor exploratory research and analysis","volume":"25","author":"Pettersen","year":"2004","journal-title":"J. Comput.\nChem."}],"container-title":["Biochemistry"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/bi5011317","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/bichaw\/article-pdf\/53\/48\/7596\/7875006\/bi5011317.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/bichaw\/article-pdf\/53\/48\/7596\/7875006\/bi5011317.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,2]],"date-time":"2026-09-02T07:26:55Z","timestamp":1788334015000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/bichaw\/article\/53\/48\/7596\/772985\/Structural-Effect-of-the-Asp345a-Insertion-in"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2014,11,18]]},"references-count":33,"journal-issue":{"issue":"48","published-online":{"date-parts":[[2014,11,18]]},"published-print":{"date-parts":[[2014,12,9]]}},"URL":"https:\/\/doi.org\/10.1021\/bi5011317","relation":{},"ISSN":["0006-2960","1520-4995"],"issn-type":[{"value":"0006-2960","type":"print"},{"value":"1520-4995","type":"electronic"}],"subject":[],"published":{"date-parts":[[2014,11,18]]}}}