{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,1]],"date-time":"2026-08-01T18:17:01Z","timestamp":1785608221000,"version":"3.56.0"},"reference-count":63,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2022,5,20]],"date-time":"2022-05-20T00:00:00Z","timestamp":1653004800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"SMART Scholarship"},{"name":"OUSD\/R&amp;E (The Under Secretary of Defense-Research and Engineering), National Defense Education Program (NDEP)\/BA-1"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Entropy"],"abstract":"<jats:p>The beta-lactamase enzyme provides effective resistance to beta-lactam antibiotics due to substrate recognition controlled by point mutations. Recently, extended-spectrum and inhibitor-resistant mutants have become a global health problem. Here, the functional dynamics that control substrate recognition in TEM beta-lactamase are investigated using all-atom molecular dynamics simulations. Comparisons are made between wild-type TEM-1 and TEM-2 and the extended-spectrum mutants TEM-10 and TEM-52, both in apo form and in complex with four different antibiotics (ampicillin, amoxicillin, cefotaxime and ceftazidime). Dynamic allostery is predicted based on a quasi-harmonic normal mode analysis using a perturbation scan. An allosteric mechanism known to inhibit enzymatic function in TEM beta-lactamase is identified, along with other allosteric binding targets. Mechanisms for substrate recognition are elucidated using multivariate comparative analysis of molecular dynamics trajectories to identify changes in dynamics resulting from point mutations and ligand binding, and the conserved dynamics, which are functionally important, are extracted as well. The results suggest that the H10-H11 loop (residues 214-221) is a secondary anchor for larger extended spectrum ligands, while the H9-H10 loop (residues 194-202) is distal from the active site and stabilizes the protein against structural changes. These secondary non-catalytically-active loops offer attractive targets for novel noncompetitive inhibitors of TEM beta-lactamase.<\/jats:p>","DOI":"10.3390\/e24050729","type":"journal-article","created":{"date-parts":[[2022,5,20]],"date-time":"2022-05-20T13:56:12Z","timestamp":1653054972000},"page":"729","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":8,"title":["Functional Dynamics of Substrate Recognition in TEM Beta-Lactamase"],"prefix":"10.3390","volume":"24","author":[{"given":"Chris","family":"Avery","sequence":"first","affiliation":[{"name":"Department of Bioinformatics and Genomics, University of North Carolina at Charlotte, Charlotte, NC 28223, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lonnie","family":"Baker","sequence":"additional","affiliation":[{"name":"Department of Bioinformatics and Genomics, University of North Carolina at Charlotte, Charlotte, NC 28223, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7711-1639","authenticated-orcid":false,"given":"Donald J.","family":"Jacobs","sequence":"additional","affiliation":[{"name":"Department of Physics and Optical Science, University of North Carolina at Charlotte, Charlotte, NC 28223, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,5,20]]},"reference":[{"key":"ref_1","unstructured":"CDC (2022, May 03). Antibiotic Resistance Threats in the United States, Available online: https:\/\/www.google.com\/url?sa=t&rct=j&q=&esrc=s&source=web&cd=&ved=2ahUKEwiHrfqY2Oj3AhUaAKYKHX6TC2kQFnoECAwQAQ&url=https%3A%2F%2Fwww.cdc.gov%2Fdrugresistance%2Fpdf%2Fthreats-report%2F2019-ar-threats-report-508.pdf&usg=AOvVaw1709sEktvf-ovR38riUreg."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"4713","DOI":"10.2147\/IDR.S290835","article-title":"Antimicrobial stewardship: Fighting antimicrobial resistance and protecting global public health","volume":"13","author":"Majumder","year":"2020","journal-title":"Infect. Drug Resist."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1113","DOI":"10.4065\/mcp.2011.0358","article-title":"Antimicrobial stewardship","volume":"Volume 86","author":"Doron","year":"2011","journal-title":"Mayo Clinic Proceedings"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"312","DOI":"10.1016\/j.envint.2018.03.044","article-title":"Antibiotic resistance in wastewater treatment plants: Tackling the black box","volume":"115","author":"Manaia","year":"2018","journal-title":"Environ. Int."},{"key":"ref_5","unstructured":"CDC (2022, May 03). Outpatient Antibiotic Prescriptions\u2014United States, Available online: https:\/\/www.cdc.gov\/antibiotic-use\/data\/report-2019.html."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"a025247","DOI":"10.1101\/cshperspect.a025247","article-title":"\u03b2-Lactams and \u03b2-lactamase inhibitors: An overview","volume":"6","author":"Bush","year":"2016","journal-title":"Cold Spring Harb. Perspect. Med."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"112829","DOI":"10.1016\/j.ejmech.2020.112829","article-title":"\u03b2-lactam antibiotics: An overview from a medicinal chemistry perspective","volume":"208","author":"Lima","year":"2020","journal-title":"Eur. J. Med. Chem."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"e01076-18","DOI":"10.1128\/AAC.01076-18","article-title":"Past and present perspectives on \u03b2-lactamases","volume":"62","author":"Bush","year":"2018","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"321","DOI":"10.1098\/rstb.1980.0049","article-title":"The structure of \u03b2-lactamases","volume":"289","author":"Ambler","year":"1980","journal-title":"Philos. Trans. R. Soc. Lond. B Biol. Sci."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"239","DOI":"10.1038\/208239a0","article-title":"Penicillinase synthesis controlled by infectious R factors in Enterobacteriaceae","volume":"208","author":"Datta","year":"1965","journal-title":"Nature"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"969","DOI":"10.1128\/AAC.01009-09","article-title":"Updated functional classification of \u03b2-lactamases","volume":"54","author":"Bush","year":"2010","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"dlab092","DOI":"10.1093\/jacamr\/dlab092","article-title":"Extended-spectrum \u03b2-lactamases: An update on their characteristics, epidemiology and detection","volume":"3","author":"Castanheira","year":"2021","journal-title":"JAC-Antimicrob. Resist."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"447","DOI":"10.1093\/jac\/43.4.447","article-title":"Inhibitor-resistant TEM \u03b2-lactamases: Phenotypic, genetic and biochemical characteristics","volume":"43","author":"Chaibi","year":"1999","journal-title":"J. Antimicrob. Chemother."},{"key":"ref_14","first-page":"e000306","article-title":"Emergence, molecular mechanisms and global spread of carbapenem-resistant Acinetobacter baumannii","volume":"5","author":"Hamidian","year":"2019","journal-title":"Microb. Genom."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"16","DOI":"10.3389\/fmolb.2018.00016","article-title":"Structural and mechanistic basis for extended-spectrum drug-resistance mutations in altering the specificity of TEM, CTX-M, and KPC \u03b2-lactamases","volume":"5","author":"Palzkill","year":"2018","journal-title":"Front. Mol. Biosci."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"127","DOI":"10.1078\/1438-4221-00198","article-title":"Molecular analysis of beta-lactamase structure and function","volume":"292","author":"Majiduddin","year":"2002","journal-title":"Int. J. Med. Microbiol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1015","DOI":"10.1111\/j.1574-6976.2010.00222.x","article-title":"Natural evolution of TEM-1 \u03b2-lactamase: Experimental reconstruction and clinical relevance","volume":"34","author":"Salverda","year":"2010","journal-title":"FEMS Microbiol. Rev."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"120","DOI":"10.1134\/S1990750817020056","article-title":"The study of the role of mutations M182T and Q39K in the TEM-72 \u03b2-lactamase structure by the molecular dynamics method","volume":"11","author":"Shcherbinin","year":"2017","journal-title":"Biochem. (Moscow) Suppl. Ser. B Biomed. Chem."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"933","DOI":"10.1128\/CMR.14.4.933-951.2001","article-title":"Extended-spectrum \u03b2-lactamases in the 21st century: Characterization, epidemiology, and detection of this important resistance threat","volume":"14","author":"Bradford","year":"2001","journal-title":"Clin. Microbiol. Rev."},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Tang, Q.Y., and Kaneko, K. (2020). Long-range correlation in protein dynamics: Confirmation by structural data and normal mode analysis. PLoS Comput. Biol., 16.","DOI":"10.1371\/journal.pcbi.1007670"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"474","DOI":"10.1038\/nchembio.98","article-title":"Allosteric regulation and catalysis emerge via a common route","volume":"4","author":"Goodey","year":"2008","journal-title":"Nat. Chem. Biol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"103","DOI":"10.1007\/BF00276625","article-title":"Allostery without conformational change","volume":"11","author":"Cooper","year":"1984","journal-title":"Eur. Biophys. J."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"5312","DOI":"10.1021\/bi992505b","article-title":"Structure-based design guides the improved efficacy of deacylation transition state analogue inhibitors of TEM-1 \u03b2-lactamase","volume":"39","author":"Ness","year":"2000","journal-title":"Biochemistry"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"238","DOI":"10.1038\/84981","article-title":"Predicting the emergence of antibiotic resistance by directed evolution and structural analysis","volume":"8","author":"Orencia","year":"2001","journal-title":"Nat. Struct. Biol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"85","DOI":"10.1016\/S0022-2836(02)00400-X","article-title":"Evolution of an antibiotic resistance enzyme constrained by stability and activity trade-offs","volume":"320","author":"Wang","year":"2002","journal-title":"J. Mol. Biol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"32149","DOI":"10.1074\/jbc.M204212200","article-title":"The structural bases of antibiotic resistance in the clinically derived mutant \u03b2-lactamases TEM-30, TEM-32, and TEM-34","volume":"277","author":"Wang","year":"2002","journal-title":"J. Biol. Chem."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"682","DOI":"10.1107\/S0907444994014496","article-title":"TEM1 \u03b2-lactamase structure solved by molecular replacement and refined structure of the S235A mutant","volume":"51","author":"Charlier","year":"1995","journal-title":"Acta Crystallogr. Sect. Biol. Crystallogr."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"33703","DOI":"10.1074\/jbc.M109.053819","article-title":"Structural and biochemical evidence that a TEM-1 \u03b2-lactamase N170G active site mutant acts via substrate-assisted catalysis","volume":"284","author":"Brown","year":"2009","journal-title":"J. Biol. Chem."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"235","DOI":"10.1093\/nar\/28.1.235","article-title":"The protein data bank","volume":"28","author":"Berman","year":"2000","journal-title":"Nucleic Acids Res."},{"key":"ref_30","unstructured":"Schr\u00f6dinger, LLC (2022, May 03). The PyMOL Molecular Graphics System, Version 1.8. Available online: https:\/\/pymol.org\/2\/."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"8617","DOI":"10.1073\/pnas.0913302107","article-title":"Structural study of TcaR and its complexes with multiple antibiotics from Staphylococcus epidermidis","volume":"107","author":"Chang","year":"2010","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"3501","DOI":"10.1016\/j.jmb.2019.07.010","article-title":"Novel and improved crystal structures of H. influenzae, E. coli and P. aeruginosa penicillin-binding protein 3 (PBP3) and N. gonorrhoeae PBP2: Toward a better understanding of \u03b2-lactam target-mediated resistance","volume":"431","author":"Bellini","year":"2019","journal-title":"J. Mol. Biol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"447","DOI":"10.1021\/bi501195g","article-title":"Molecular basis for the catalytic specificity of the CTX-M extended-spectrum \u03b2-lactamases","volume":"54","author":"Adamski","year":"2015","journal-title":"Biochemistry"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"3443","DOI":"10.1021\/acs.biochem.7b00176","article-title":"The drug-resistant variant P167S expands the substrate profile of CTX-M \u03b2-lactamases for oxyimino-cephalosporin antibiotics by enlarging the active site upon acylation","volume":"56","author":"Patel","year":"2017","journal-title":"Biochemistry"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"17","DOI":"10.1186\/1758-2946-4-17","article-title":"Avogadro: An advanced semantic chemical editor, visualization, and analysis platform","volume":"4","author":"Hanwell","year":"2012","journal-title":"J. Cheminf."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"148","DOI":"10.1038\/s41597-022-01261-1","article-title":"AB-DB: Force-Field parameters, MD trajectories, QM-based data, and Descriptors of Antimicrobials","volume":"9","author":"Gervasoni","year":"2022","journal-title":"Sci. Data"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"455","DOI":"10.1002\/jcc.21334","article-title":"AutoDock Vina: Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading","volume":"31","author":"Trott","year":"2010","journal-title":"J. Comput. Chem."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"3198","DOI":"10.1038\/s41598-021-82410-1","article-title":"New machine learning and physics-based scoring functions for drug discovery","volume":"11","author":"Guedes","year":"2021","journal-title":"Sci. Rep."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"667","DOI":"10.1021\/acs.jcim.9b00905","article-title":"Highly flexible ligand docking: Benchmarking of the DockThor program on the LEADS-PEP protein\u2013peptide data set","volume":"60","author":"Santos","year":"2020","journal-title":"J. Chem. Inf. Model."},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"Farmer, J., Kanwal, F., Nikulsin, N., Tsilimigras, M.C., and Jacobs, D.J. (2017). Statistical measures to quantify similarity between molecular dynamics simulation trajectories. Entropy, 19.","DOI":"10.3390\/e19120646"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"1950","DOI":"10.1002\/prot.22711","article-title":"Improved side-chain torsion potentials for the Amber ff99SB protein force field","volume":"78","author":"Piana","year":"2010","journal-title":"Proteins Struct. Funct. Bioinform."},{"key":"ref_42","first-page":"U403","article-title":"Antechamber: An accessory software package for molecular mechanical calculations","volume":"222","author":"Wang","year":"2001","journal-title":"J. Am. Chem. Soc"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"367","DOI":"10.1186\/1756-0500-5-367","article-title":"ACPYPE-Antechamber python parser interface","volume":"5","author":"Vranken","year":"2012","journal-title":"BMC Res. Notes"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"19","DOI":"10.1016\/j.softx.2015.06.001","article-title":"GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers","volume":"1","author":"Abraham","year":"2015","journal-title":"SoftwareX"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"226","DOI":"10.1186\/s12859-021-04140-5","article-title":"JEDi: Java essential dynamics inspector\u2014A molecular trajectory analysis toolkit","volume":"22","author":"David","year":"2021","journal-title":"BMC Bioinform."},{"key":"ref_46","doi-asserted-by":"crossref","unstructured":"Farmer, J., and Jacobs, D. (2018). High throughput nonparametric probability density estimation. PLoS ONE, 13.","DOI":"10.1371\/journal.pone.0196937"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"101017","DOI":"10.1016\/j.softx.2022.101017","article-title":"MATLAB tool for probability density assessment and nonparametric estimation","volume":"18","author":"Farmer","year":"2022","journal-title":"SoftwareX"},{"key":"ref_48","first-page":"1099","article-title":"Quasi-harmonic method for studying very low frequency modes in proteins","volume":"23","author":"Levy","year":"1984","journal-title":"Biopolym. Orig. Res. Biomol."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"17508","DOI":"10.1021\/jacs.7b09268","article-title":"Decomposing dynamical couplings in mutated scFv antibody fragments into stabilizing and destabilizing effects","volume":"139","author":"Uddin","year":"2017","journal-title":"J. Am. Chem. Soc."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"4247","DOI":"10.1038\/s41598-021-83269-y","article-title":"Molecular function recognition by supervised projection pursuit machine learning","volume":"11","author":"Grear","year":"2021","journal-title":"Sci. Rep."},{"key":"ref_51","first-page":"221","article-title":"Biased Hypothesis Formation From Projection Pursuit","volume":"1","author":"Patterson","year":"2021","journal-title":"Adv. Artif. Intell. Mach. Learn."},{"key":"ref_52","unstructured":"(2022, May 03). SPLOC GitHub. Available online: https:\/\/github.com\/BioMolecularPhysicsGroup-UNCC\/MachineLearning\/tree\/master\/SPLOC."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"1283","DOI":"10.1016\/j.jmb.2003.12.068","article-title":"Allosteric inhibition through core disruption","volume":"336","author":"Horn","year":"2004","journal-title":"J. Mol. Biol."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"29","DOI":"10.1002\/prot.340140106","article-title":"Probing \u03b2-lactamase structure and function using random replacement mutagenesis","volume":"14","author":"Palzkill","year":"1992","journal-title":"Proteins Struct. Funct. Bioinform."},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"238","DOI":"10.1016\/j.jmb.2008.07.082","article-title":"Increased folding stability of TEM-1 \u03b2-lactamase by in vitro selection","volume":"383","author":"Kather","year":"2008","journal-title":"J. Mol. Biol."},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"151","DOI":"10.1016\/j.jmb.2008.09.009","article-title":"Genetic and structural characterization of an L201P global suppressor substitution in TEM-1 \u03b2-lactamase","volume":"384","author":"Marciano","year":"2008","journal-title":"J. Mol. Biol."},{"key":"ref_57","doi-asserted-by":"crossref","first-page":"e66567","DOI":"10.7554\/eLife.66567","article-title":"Allosteric communication in class A beta-lactamases occurs via cooperative coupling of loop dynamics","volume":"10","author":"Galdadas","year":"2021","journal-title":"eLife"},{"key":"ref_58","doi-asserted-by":"crossref","first-page":"412","DOI":"10.1002\/prot.340170408","article-title":"Essential dynamics of proteins","volume":"17","author":"Amadei","year":"1993","journal-title":"Proteins Struct. Funct. Bioinform."},{"key":"ref_59","doi-asserted-by":"crossref","unstructured":"David, C.C., and Jacobs, D.J. (2014). Principal component analysis: A method for determining the essential dynamics of proteins. Protein Dynamics, Springer.","DOI":"10.1007\/978-1-62703-658-0_11"},{"key":"ref_60","doi-asserted-by":"crossref","first-page":"3579","DOI":"10.1128\/AAC.48.9.3579-3582.2004","article-title":"Biochemical characterization of laboratory mutants of extended-spectrum \u03b2-lactamase TEM-60","volume":"48","author":"Caporale","year":"2004","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_61","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1080\/14756366.2016.1201813","article-title":"An evolutionarily conserved allosteric site modulates beta-lactamase activity","volume":"31","author":"Avci","year":"2016","journal-title":"J. Enzym. Inhib. Med. Chem."},{"key":"ref_62","doi-asserted-by":"crossref","first-page":"46295","DOI":"10.1074\/jbc.M407606200","article-title":"Site-saturation mutagenesis of Tyr-105 reveals its importance in substrate stabilization and discrimination in TEM-1 \u03b2-lactamase","volume":"279","author":"Doucet","year":"2004","journal-title":"J. Biol. Chem."},{"key":"ref_63","doi-asserted-by":"crossref","first-page":"21448","DOI":"10.1074\/jbc.M609777200","article-title":"NMR investigation of Tyr105 mutants in TEM-1 \u03b2-lactamase: Dynamics are correlated with function","volume":"282","author":"Doucet","year":"2007","journal-title":"J. Biol. Chem."}],"container-title":["Entropy"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1099-4300\/24\/5\/729\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T23:15:30Z","timestamp":1760138130000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1099-4300\/24\/5\/729"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,5,20]]},"references-count":63,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2022,5]]}},"alternative-id":["e24050729"],"URL":"https:\/\/doi.org\/10.3390\/e24050729","relation":{},"ISSN":["1099-4300"],"issn-type":[{"value":"1099-4300","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,5,20]]}}}