{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,30]],"date-time":"2026-06-30T01:01:12Z","timestamp":1782781272149,"version":"3.54.5"},"reference-count":57,"publisher":"MDPI AG","issue":"13","license":[{"start":{"date-parts":[[2023,6,30]],"date-time":"2023-06-30T00:00:00Z","timestamp":1688083200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["21973004"],"award-info":[{"award-number":["21973004"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Alligator sinensis cathelicidins (As-CATHs) are antimicrobial peptides extracted from alligators that enable alligators to cope with diseases caused by bacterial infections. This study assessed the damaging effects of sequence-truncated and residue-substituted variants of As-CATH4, AS4-1, AS4-5, and AS4-9 (with decreasing charges but increasing hydrophobicity) on the membranes of Gram-negative bacteria at the molecular level by using coarse-grained molecular dynamics simulations. The simulations predicted that all the variants disrupt the structures of the inner membrane of Gram-negative bacteria, with AS4-9 having the highest antibacterial activity that is able to squeeze the membrane and extract lipids from the membrane. However, none of them can disrupt the structure of asymmetric outer membrane of Gram-negative bacteria, which is composed of lipopolysaccharides in the outer leaflet and phospholipids in the inner leaflet. Nonetheless, the adsorption of AS4-9 induces lipid scrambling in the membrane by lowering the free energy of a phospholipid flipping from the inner leaflet up to the outer leaflet. Upon binding onto the lipid-scrambled outer membrane, AS4-9s are predicted to squeeze and extract phospholipids from the membrane, AS4-5s have a weak pull-out effect, and AS4-1s mainly stay free in water without any lipid-extracting function. These findings provide inspiration for the development of potent therapeutic agents targeting bacteria.<\/jats:p>","DOI":"10.3390\/ijms241310962","type":"journal-article","created":{"date-parts":[[2023,6,30]],"date-time":"2023-06-30T16:06:37Z","timestamp":1688141197000},"page":"10962","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["How Alligator Immune Peptides Kill Gram-Negative Bacteria: A Lipid-Scrambling, Squeezing, and Extracting Mechanism Revealed by Theoretical Simulations"],"prefix":"10.3390","volume":"24","author":[{"given":"Xiangyuan","family":"Li","sequence":"first","affiliation":[{"name":"Key Laboratory of Theoretical and Computational Photochemistry, Ministry of Education, College of Chemistry, Beijing Normal University, Beijing 100875, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lei","family":"Fu","sequence":"additional","affiliation":[{"name":"Key Laboratory of Theoretical and Computational Photochemistry, Ministry of Education, College of Chemistry, Beijing Normal University, Beijing 100875, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shan","family":"Zhang","sequence":"additional","affiliation":[{"name":"Key Laboratory of Theoretical and Computational Photochemistry, Ministry of Education, College of Chemistry, Beijing Normal University, Beijing 100875, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yipeng","family":"Wang","sequence":"additional","affiliation":[{"name":"Department of Biopharmaceutical Sciences, College of Pharmaceutical Sciences, Soochow University, Suzhou 215123, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4414-3671","authenticated-orcid":false,"given":"Lianghui","family":"Gao","sequence":"additional","affiliation":[{"name":"Key Laboratory of Theoretical and Computational Photochemistry, Ministry of Education, College of Chemistry, Beijing Normal University, Beijing 100875, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2023,6,30]]},"reference":[{"key":"ref_1","first-page":"801","article-title":"Human Antimicrobial Peptides: Spectrum, Mode of Action and Resistance Mechanisms","volume":"27","author":"Seyedi","year":"2020","journal-title":"Int. J. Pept. Res. Ther."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"171","DOI":"10.1146\/annurev-physchem-103019-033434","article-title":"Molecular Simulations of Gram-Negative Bacterial Membranes Come of Age","volume":"71","author":"Im","year":"2020","journal-title":"Annu. Rev. Phys. Chem."},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Dash, R., and Bhattacharjya, S. (2021). Thanatin: An Emerging Host Defense Antimicrobial Peptide with Multiple Modes of Action. Int. J. Mol. Sci., 22.","DOI":"10.3390\/ijms22041522"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"37","DOI":"10.1038\/nrd3591","article-title":"Designing antimicrobial peptides: Form follows function","volume":"11","author":"Fjell","year":"2011","journal-title":"Nat. Rev. Drug Discov."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"389","DOI":"10.1038\/415389a","article-title":"Antimicrobial peptides of multicellular organisms","volume":"415","author":"Zasloff","year":"2002","journal-title":"Nature"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"582779","DOI":"10.3389\/fmicb.2020.582779","article-title":"Antimicrobial Peptides: Classification, Design, Application and Research Progress in Multiple Fields","volume":"11","author":"Huan","year":"2020","journal-title":"Front. Microbiol."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Guryanova, S.V., and Ovchinnikova, T.V. (2022). Immunomodulatory and Allergenic Properties of Antimicrobial Peptides. Int. J. Mol. Sci., 23.","DOI":"10.3390\/ijms23052499"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1337","DOI":"10.1038\/ncomms2317","article-title":"Extensive diversification of IgH subclass-encoding genes and IgM subclass switching in crocodilians","volume":"4","author":"Cheng","year":"2013","journal-title":"Nat. Commun."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1091","DOI":"10.1038\/cr.2013.104","article-title":"Genome analysis and signature discovery for diving and sensory properties of the endangered Chinese alligator","volume":"23","author":"Wan","year":"2013","journal-title":"Cell Res."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"133","DOI":"10.1016\/j.cbpb.2005.10.005","article-title":"Comparisons of innate immune activity of all known living crocodylian species","volume":"143","author":"Merchant","year":"2006","journal-title":"Comp. Biochem. Physiol. B Biochem. Mol. Biol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"221","DOI":"10.1016\/j.vetimm.2005.10.001","article-title":"Broad spectrum antimicrobial activity of leukocyte extracts from the American alligator (Alligator mississippiensis)","volume":"110","author":"Merchant","year":"2006","journal-title":"Vet. Immunol. Immunopathol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"2861","DOI":"10.1042\/BCJ20170334","article-title":"As-CATH1-6, novel cathelicidins with potent antimicrobial and immunomodulatory properties from Alligator sinensis, play pivotal roles in host antimicrobial immune responses","volume":"474","author":"Chen","year":"2017","journal-title":"Biochem. J."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"14701","DOI":"10.1021\/acs.jmedchem.2c01238","article-title":"Hydrophobicity Determines the Bacterial Killing Rate of alpha-Helical Antimicrobial Peptides and Influences the Bacterial Resistance Development","volume":"65","author":"Zhang","year":"2022","journal-title":"J. Med. Chem."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"35714","DOI":"10.1074\/jbc.M104925200","article-title":"Interaction of cationic antimicrobial peptides with model membranes","volume":"276","author":"Zhang","year":"2001","journal-title":"J. Biol. Chem."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"2841","DOI":"10.3168\/jds.S0022-0302(98)75843-6","article-title":"Properties of a heparin-binding peptide derived from bovine lactoferrin","volume":"81","author":"Shimazaki","year":"1998","journal-title":"J. Dairy Sci."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"29","DOI":"10.1016\/S0005-2736(99)00199-6","article-title":"Interaction of antimicrobial peptides with biological and model membranes: Structural and charge requirements for activity","volume":"1462","author":"Sitaram","year":"1999","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"15739","DOI":"10.1039\/c2cp43099a","article-title":"The antimicrobial peptide aurein 1.2 disrupts model membranes via the carpet mechanism","volume":"14","author":"Fernandez","year":"2012","journal-title":"Phys. Chem. Chem. Phys."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"786","DOI":"10.1038\/s41579-021-00585-w","article-title":"Antibiofilm activity of host defence peptides: Complexity provides opportunities","volume":"19","author":"Hancock","year":"2021","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"3486","DOI":"10.1016\/j.bpj.2022.08.007","article-title":"Polymyxins induce lipid scrambling and disrupt the homeostasis of Gram-negative bacteria membrane","volume":"121","author":"Fu","year":"2022","journal-title":"Biophys. J."},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Barreto-Santamaria, A., Arevalo-Pinzon, G., Patarroyo, M.A., and Patarroyo, M.E. (2021). How to Combat Gram-Negative Bacteria Using Antimicrobial Peptides: A Challenge or an Unattainable Goal?. Antibiotics, 10.","DOI":"10.3390\/antibiotics10121499"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1518","DOI":"10.1016\/j.bpj.2018.08.040","article-title":"Peptide-Lipid Interaction Sites Affect Vesicles\u2019 Responses to Antimicrobial Peptides","volume":"115","author":"Shi","year":"2018","journal-title":"Biophys. J."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"850","DOI":"10.1021\/jp505497k","article-title":"Theoretical insight into the relationship between the structures of antimicrobial peptides and their actions on bacterial membranes","volume":"119","author":"Chen","year":"2015","journal-title":"J. Phys. Chem. B"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"13233","DOI":"10.1021\/acs.langmuir.9b02175","article-title":"Peripheral Antimicrobial Peptide Gomesin Induces Membrane Protrusion, Folding, and Laceration","volume":"35","author":"Zhang","year":"2019","journal-title":"Langmuir"},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Zhang, S., Ma, M., Shao, Z., Zhang, J., Fu, L., Li, X., Fang, W., and Gao, L. (2021). Structure and Formation Mechanism of Antimicrobial Peptides Temporin B- and L-Induced Tubular Membrane Protrusion. Int. J. Mol. Sci., 22.","DOI":"10.3390\/ijms222011015"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"131","DOI":"10.1016\/j.actbio.2022.04.025","article-title":"Anisaxins, helical antimicrobial peptides from marine parasites, kill resistant bacteria by lipid extraction and membrane disruption","volume":"146","author":"Roncevic","year":"2022","journal-title":"Acta Biomater."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"6184","DOI":"10.1021\/acs.chemrev.8b00460","article-title":"Computational Modeling of Realistic Cell Membranes","volume":"119","author":"Marrink","year":"2019","journal-title":"Chem. Rev."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"227","DOI":"10.1016\/j.physb.2018.02.043","article-title":"An overview of neutron scattering and molecular dynamics simulation studies of phospholipid bilayers in room-temperature ionic liquid\/water solutions","volume":"551","author":"Benedetto","year":"2018","journal-title":"Phys. B Condens. Matter"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"E8518","DOI":"10.1073\/pnas.1806714115","article-title":"Phospholipid retention in the absence of asymmetry strengthens the outer membrane permeability barrier to last-resort antibiotics","volume":"115","author":"Powers","year":"2018","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"187","DOI":"10.1038\/s41589-020-00694-2","article-title":"Dynamics of an LPS translocon induced by substrate and an antimicrobial peptide","volume":"17","author":"Fiorentino","year":"2021","journal-title":"Nat. Chem. Biol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"E1565","DOI":"10.1073\/pnas.1601375113","article-title":"Disruption of lipid homeostasis in the Gram-negative cell envelope activates a novel cell death pathway","volume":"113","author":"Sutterlin","year":"2016","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"348","DOI":"10.1038\/d41586-020-02256-x","article-title":"Structure of a lipopolysaccharide regulator reveals a road to new antibiotics","volume":"584","author":"Bishop","year":"2020","journal-title":"Nature"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"705","DOI":"10.1146\/annurev-biochem-040320-105145","article-title":"Structure and Mechanism of the Lipid Flippase MurJ","volume":"91","author":"Kuk","year":"2022","journal-title":"Annu. Rev. Biochem."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"233","DOI":"10.1038\/nature23649","article-title":"Structural basis of MsbA-mediated lipopolysaccharide transport","volume":"549","author":"Mi","year":"2017","journal-title":"Nature"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"371","DOI":"10.1038\/s41586-022-04555-x","article-title":"Structural basis of lipopolysaccharide maturation by the O-antigen ligase","volume":"604","author":"Ashraf","year":"2022","journal-title":"Nature"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1605","DOI":"10.1002\/jcc.20084","article-title":"UCSF Chimera\u2014A visualization system for exploratory research and analysis","volume":"25","author":"Pettersen","year":"2004","journal-title":"J. Comput. Chem."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"325","DOI":"10.1021\/ct900487a","article-title":"On the Validation of Molecular Dynamics Simulations of Saturated and cis-Monounsaturated Phosphatidylcholine Lipid Bilayers: A Comparison with Experiment","volume":"6","author":"Poger","year":"2010","journal-title":"J. Chem. Theory Comput."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"306","DOI":"10.1007\/s008940100045","article-title":"GROMACS 3.0: A package for molecular simulation and trajectory analysis","volume":"7","author":"Lindahl","year":"2001","journal-title":"J. Mol. Mode"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"1701","DOI":"10.1002\/jcc.20291","article-title":"GROMACS: Fast, flexible, and free","volume":"26","author":"Lindahl","year":"2005","journal-title":"J. Comput. Chem."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"435","DOI":"10.1021\/ct700301q","article-title":"GROMACS 4: Algorithms for Highly Efficient, Load-Balanced, and Scalable Molecular Simulation","volume":"4","author":"Hess","year":"2008","journal-title":"J. Chem. Theory Comput."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"845","DOI":"10.1093\/bioinformatics\/btt055","article-title":"GROMACS 4.5: A high-throughput and highly parallel open source molecular simulation toolkit","volume":"29","author":"Pronk","year":"2013","journal-title":"Bioinformatics"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"134101","DOI":"10.1063\/1.2013227","article-title":"A configurational temperature Nose-Hoover thermostat","volume":"123","author":"Braga","year":"2005","journal-title":"J. Chem. Phys."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"234505","DOI":"10.1063\/1.2121687","article-title":"A general purpose model for the condensed phases of water: TIP4P\/2005","volume":"123","author":"Abascal","year":"2005","journal-title":"J. Chem. Phys."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"1055","DOI":"10.1080\/00268978300102851","article-title":"Constant pressure molecular dynamics for molecular systems","volume":"50","author":"Klein","year":"1983","journal-title":"Mol. Phys."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"1695","DOI":"10.1103\/PhysRevA.31.1695","article-title":"Canonical dynamics: Equilibrium phase-space distributions","volume":"31","author":"Hoover","year":"1985","journal-title":"Phys. Rev. A"},{"key":"ref_45","first-page":"255","article-title":"A molecular dynamics method for simulations in the canonical ensemble","volume":"52","year":"2006","journal-title":"Mol. Phys."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"750","DOI":"10.1021\/jp036508g","article-title":"Coarse grained model for semiquantitative lipid simulations","volume":"108","author":"Marrink","year":"2004","journal-title":"J. Phys. Chem. B"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"7812","DOI":"10.1021\/jp071097f","article-title":"The MARTINI force field: Coarse grained model for biomolecular simulations","volume":"111","author":"Marrink","year":"2007","journal-title":"J. Phys. Chem. B"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"819","DOI":"10.1021\/ct700324x","article-title":"The MARTINI Coarse-Grained Force Field: Extension to Proteins","volume":"4","author":"Monticelli","year":"2008","journal-title":"J. Chem. Theory Comput."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"687","DOI":"10.1021\/ct300646g","article-title":"Improved Parameters for the Martini Coarse-Grained Protein Force Field","volume":"9","author":"Singh","year":"2013","journal-title":"J. Chem. Theory Comput."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"111","DOI":"10.1007\/s12551-021-00913-7","article-title":"Model architectures for bacterial membranes","volume":"14","author":"Carey","year":"2022","journal-title":"Biophys. Rev."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"57","DOI":"10.1038\/nrmicro1322","article-title":"Advances in understanding bacterial outer-membrane biogenesis","volume":"4","author":"Ruiz","year":"2006","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"1451","DOI":"10.1016\/j.bbalip.2016.10.006","article-title":"Lipopolysaccharide biogenesis and transport at the outer membrane of Gram-negative bacteria","volume":"1862","author":"Sperandeo","year":"2017","journal-title":"Biochim. Biophys. Acta Mol. Cell Biol. Lipids"},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"2144","DOI":"10.1021\/acs.jctc.5b00209","article-title":"Computational Lipidomics with insane: A Versatile Tool for Generating Custom Membranes for Molecular Simulations","volume":"11","author":"Wassenaar","year":"2015","journal-title":"J. Chem. Theory Comput."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"1672","DOI":"10.1021\/acs.biochem.6b01063","article-title":"Through the Lipopolysaccharide Glass: A Potent Antimicrobial Peptide Induces Phase Changes in Membranes","volume":"56","author":"Jefferies","year":"2017","journal-title":"Biochemistry"},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"635","DOI":"10.1146\/annurev.biochem.71.110601.135414","article-title":"Lipopolysaccharide endotoxins","volume":"71","author":"Raetz","year":"2002","journal-title":"Annu. Rev. Biochem."},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"11170","DOI":"10.1021\/acs.jpcb.6b06615","article-title":"Molecular Dynamics Simulations Predict the Pathways via Which Pristine Fullerenes Penetrate Bacterial Membranes","volume":"120","author":"Hsu","year":"2016","journal-title":"J. Phys. Chem. B"},{"key":"ref_57","doi-asserted-by":"crossref","first-page":"3713","DOI":"10.1021\/ct100494z","article-title":"g_wham\u2014A Free Weighted Histogram Analysis Implementation Including Robust Error and Autocorrelation Estimates","volume":"6","author":"Hub","year":"2010","journal-title":"J. Chem. Theory Comput."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/24\/13\/10962\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T20:04:07Z","timestamp":1760126647000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/24\/13\/10962"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,6,30]]},"references-count":57,"journal-issue":{"issue":"13","published-online":{"date-parts":[[2023,7]]}},"alternative-id":["ijms241310962"],"URL":"https:\/\/doi.org\/10.3390\/ijms241310962","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,6,30]]}}}