{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,10]],"date-time":"2026-03-10T07:12:26Z","timestamp":1773126746549,"version":"3.50.1"},"reference-count":40,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2023,1,29]],"date-time":"2023-01-29T00:00:00Z","timestamp":1674950400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National Research Council of Thailand (NRCT)","award":["NRCT5-RSA63002-12"],"award-info":[{"award-number":["NRCT5-RSA63002-12"]}]},{"name":"Kasetsart University (Bangkok, Thailand)","award":["NRCT5-RSA63002-12"],"award-info":[{"award-number":["NRCT5-RSA63002-12"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Toxins"],"abstract":"<jats:p>Receptor binding is a prerequisite process to exert the mosquitocidal activity of the Cry4Ba toxin of Bacillus thuringiensis subsp. israelensis. The beta-sheet prism (domain II) and beta-sheet sandwich (domain III) of the Cry4Ba toxin have been implicated in receptor binding, albeit the precise binding mechanisms of these remain unclear. In this work, alanine scanning was used to determine the contribution to receptor binding of some aromatic and hydrophobic residues on the surface of domains II and III that are predicted to be responsible for binding to the Aedes aegypti membrane-bound alkaline phosphatase (Aa-mALP) receptor. Larvicidal activity assays against A. aegypti larvae revealed that aromatic residues (Trp327 on the \u03b22 strand, Tyr347 on the \u03b23\u2013\u03b24 loop, and Tyr359 on the \u03b24 strand) of domain II were important to the toxicity of the Cry4Ba toxin. Quantitative binding assays using enzyme-linked immunosorbent assay (ELISA) showed similar decreasing trends in binding to the Aa-mALP receptor and in toxicity of the Cry4Ba mutants Trp327Ala, Tyr347Ala, and Tyr359Ala, suggesting that a possible function of these surface-exposed aromatic residues is receptor binding. In addition, binding assays of the Cry4Ba toxin to the mutants of the binding residues Gly513, Ser490, and Phe497 of the Aa-mALP receptor supported the binding function of Trp327, Tyr347, and Tyr359 of the Cry4Ba toxin, respectively. Altogether, our results showed for the first time that aromatic residues on a side surface of the Cry4Ba domain II function in receptor binding. This finding provides greater insight into the possible molecular mechanisms of the Cry4Ba toxin.<\/jats:p>","DOI":"10.3390\/toxins15020114","type":"journal-article","created":{"date-parts":[[2023,1,30]],"date-time":"2023-01-30T01:33:37Z","timestamp":1675042417000},"page":"114","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Aromatic Residues on the Side Surface of Cry4Ba-Domain II of Bacillus\u00a0thuringiensis subsp. israelensis Function in Binding to Their Counterpart Residues on the Aedes aegypti Alkaline Phosphatase Receptor"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0453-3464","authenticated-orcid":false,"given":"Anon","family":"Thammasittirong","sequence":"first","affiliation":[{"name":"Department of Microbiology, Faculty of Liberal Arts and Science, Kasetsart University, Nakhon Pathom 73140, Thailand"},{"name":"Microbial Biotechnology Unit, Faculty of Liberal Arts and Science, Kasetsart University, Nakhon Pathom 73140, Thailand"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3643-3030","authenticated-orcid":false,"given":"Sutticha Na-Ranong","family":"Thammasittirong","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Faculty of Liberal Arts and Science, Kasetsart University, Nakhon Pathom 73140, Thailand"},{"name":"Microbial Biotechnology Unit, Faculty of Liberal Arts and Science, Kasetsart University, Nakhon Pathom 73140, Thailand"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2023,1,29]]},"reference":[{"key":"ref_1","unstructured":"WHO (2009). Dengue Guidelines for Diagnosis, Treatment, Prevention and Control: New Edition, WHO."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"17","DOI":"10.1007\/s00284-020-02284-w","article-title":"Current understanding of the pathogenesis of dengue virus infection","volume":"78","author":"Bhatt","year":"2021","journal-title":"Curr. Microbiol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"9","DOI":"10.7861\/clinmed.2021-0791","article-title":"Dengue infection: Global importance, immunopathology and management","volume":"22","author":"Kularatne","year":"2022","journal-title":"Clin. Med."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"504","DOI":"10.1038\/nature12060","article-title":"The global distribution and burden of dengue","volume":"496","author":"Bhatt","year":"2013","journal-title":"Nature"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1508","DOI":"10.1038\/s41564-019-0476-8","article-title":"The current and future global distribution and population at risk of dengue","volume":"4","author":"Messina","year":"2019","journal-title":"Nat. Microbiol."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"WHO (2019). Dengue vaccine: WHO position paper, September 2018-Recommendations. Vaccine, 37, 4848\u20134849.","DOI":"10.1016\/j.vaccine.2018.09.063"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"247","DOI":"10.1007\/s12275-022-1625-y","article-title":"Current status and perspectives on vaccine development against dengue virus infection","volume":"60","author":"Park","year":"2022","journal-title":"J. Microbiol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"133","DOI":"10.2987\/8756-971X(2007)23[133:BTSIAB]2.0.CO;2","article-title":"Bacillus thuringiensis serovariety israelensis and Bacillus sphaericus for mosquito control","volume":"23","author":"Lawrence","year":"2007","journal-title":"J. Am. Mosq. Control Assoc."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Silva-Filha, M., Rom\u00e3o, T., Rezende, T., Carvalho, K., de Menezes, H.G., Nascimento, N.A.D., Sober\u00f3n, M., and Bravo, A. (2021). Bacterial toxins active against mosquitoes: Mode of action and resistance. Toxins, 13.","DOI":"10.3390\/toxins13080523"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"894","DOI":"10.1590\/0074-0276130155","article-title":"Monitoring resistance to Bacillus thuringiensis subsp. israelensis in the field by performing bioassays with each Cry toxin separately","volume":"108","author":"Tetreau","year":"2013","journal-title":"Mem. Inst. Oswaldo Cruz"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"137800","DOI":"10.1016\/j.scitotenv.2020.137800","article-title":"Environmental and socioeconomic effects of mosquito control in Europe using the biocide Bacillus thuringiensis subsp. israelensis (Bti)","volume":"724","author":"Patil","year":"2020","journal-title":"Sci. Total Environ."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"673","DOI":"10.1186\/s13071-018-3246-1","article-title":"Long-term exposure of Aedes aegypti to Bacillus thuringiensis svar. israelensis did not involve altered susceptibility to this microbial larvicide or to other control agents","volume":"11","author":"Carvalho","year":"2018","journal-title":"Parasites Vectors"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1222","DOI":"10.3390\/toxins6041222","article-title":"Bacillus thuringiensis subsp. israelensis and its dipteran-specific toxins","volume":"6","year":"2014","journal-title":"Toxins"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"363","DOI":"10.1016\/j.jmb.2005.02.013","article-title":"Crystal structure of the mosquito-larvicidal toxin Cry4Ba and its biological implications","volume":"348","author":"Boonserm","year":"2005","journal-title":"J. Mol. Biol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"279","DOI":"10.1016\/j.ibmb.2009.01.002","article-title":"Proteomic identification of Bacillus thuringiensis subsp. israelensis toxin Cry4Ba binding proteins in midgut membranes from Aedes (Stegomyia) aegypti Linnaeus (Diptera, Culicidae) larvae","volume":"39","author":"Bayyareddy","year":"2009","journal-title":"Insect Biochem. Mol. Biol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"683","DOI":"10.1016\/j.ibmb.2012.06.001","article-title":"Aedes aegypti alkaline phosphatase ALP1 is a functional receptor of Bacillus thuringiensis Cry4Ba and Cry11Aa toxins","volume":"42","author":"Reyes","year":"2012","journal-title":"Insect Biochem. Mol. Biol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"708","DOI":"10.1016\/j.bbrc.2011.03.085","article-title":"In vivo identification of Bacillus thuringiensis Cry4Ba toxin receptors by RNA interference knockdown of glycosylphosphatidylinositol-linked aminopeptidase N transcripts in Aedes aegypti larvae","volume":"407","author":"Saengwiman","year":"2011","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"159","DOI":"10.1016\/j.ibmb.2010.11.006","article-title":"Functional expression in insect cells of glycosylphosphatidylinositol-linked alkaline phosphatase from Aedes aegypti larval midgut: A Bacillus thuringiensis Cry4Ba toxin receptor","volume":"41","author":"Dechklar","year":"2011","journal-title":"Insect Biochem. Mol. Biol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"300","DOI":"10.1016\/j.bbrc.2015.04.026","article-title":"Two specific membrane-bound aminopeptidase N isoforms from Aedes aegypti larvae serve as functional receptors for the Bacillus thuringiensis Cry4Ba toxin implicating counterpart specificity","volume":"461","author":"Aroonkesorn","year":"2015","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"6836","DOI":"10.1128\/AEM.05775-11","article-title":"Aedes aegypti membrane-bound alkaline phosphatase expressed in Escherichia coli retains high-affinity binding for Bacillus thuringiensis Cry4Ba toxin","volume":"77","author":"Thammasittirong","year":"2011","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Thammasittirong, A., Thammasittirong, S.N.-R., Imtong, C., Charoenjotivadhanakul, S., Sakdee, S., Li, H.-C., Okonogi, S., and Angsuthanasombat, C. (2021). Bacillus thuringiensis Cry4Ba insecticidal toxin exploits Leu615 in its C-terminal domain to interact with a target receptor\u2014Aedes aegypti membrane-bound alkaline phosphatase. Toxins, 13.","DOI":"10.3390\/toxins13080553"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"139","DOI":"10.1111\/j.1574-6968.2009.01774.x","article-title":"Correlative effect on the toxicity of three surface-exposed loops in the receptor-binding domain of the Bacillus thuringiensis Cry4Ba toxin","volume":"300","author":"Khaokhiew","year":"2009","journal-title":"FEMS Microbiol. Lett."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1016\/0263-7855(96)00018-5","article-title":"VMD: Visual molecular dynamics","volume":"14","author":"Humphrey","year":"1996","journal-title":"J. Mol. Graph."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"W333","DOI":"10.1093\/nar\/gkt450","article-title":"BeAtMuSiC: Prediction of changes in protein\u2013protein binding affinity on mutations","volume":"41","author":"Dehouck","year":"2013","journal-title":"Nucleic Acids Res."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"317","DOI":"10.3109\/07388551.2014.960793","article-title":"Bacillus thuringiensis: Mechanism of action, resistance, and new applications: A review","volume":"36","author":"Melo","year":"2016","journal-title":"Crit. Rev. Biotechnol."},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"V\u00edlchez, S. (2020). Making 3D-Cry toxin mutants: Much more than a tool of understanding toxins mechanism of action. Toxins, 12.","DOI":"10.3390\/toxins12090600"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1255","DOI":"10.2174\/138161212799436412","article-title":"Computational prediction of protein hot spot residues","volume":"18","author":"Morrow","year":"2012","journal-title":"Curr. Pharm. Des."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"539","DOI":"10.1002\/prot.10164","article-title":"Monte Carlo simulations of the peptide recognition at the consensus binding site of the constant fragment of human immunoglobulin G: The energy landscape analysis of a hot spot at the intermolecular interface","volume":"48","author":"Verkhivker","year":"2002","journal-title":"Proteins"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"13713","DOI":"10.18632\/oncotarget.24459","article-title":"Computational modeling suggests impaired interactions between NKX2.5 and GATA4 in individuals carrying a novel pathogenic D16N NKX2.5 mutation","volume":"9","author":"Mattapally","year":"2018","journal-title":"Oncotarget"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"2442","DOI":"10.1128\/aem.59.8.2442-2448.1993","article-title":"Structural stability of Bacillus thuringiensis delta-endotoxin homolog-scanning mutants determined by susceptibility to proteases","volume":"59","author":"Almond","year":"1993","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"5772","DOI":"10.1073\/pnas.1030237100","article-title":"Protein-protein interactions: Structurally conserved residues distinguish between binding sites and exposed protein surfaces","volume":"100","author":"Ma","year":"2003","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"e2693","DOI":"10.1002\/jmr.2693","article-title":"Local and global anatomy of antibody-protein antigen recognition","volume":"31","author":"Wang","year":"2018","journal-title":"J. Mol. Recognit."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"E2656","DOI":"10.1073\/pnas.1401131111","article-title":"Origins of specificity and affinity in antibody\u2013protein interactions","volume":"111","author":"Peng","year":"2014","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"2812","DOI":"10.1128\/IAI.00126-08","article-title":"Role of aromatic amino acids in receptor binding activity and subunit assembly of the cytolethal distending toxin of Aggregatibacter actinomycetemcomitans","volume":"76","author":"Cao","year":"2008","journal-title":"Infect. Immun."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"14347","DOI":"10.1021\/bi061334p","article-title":"Identification of functional domains of Clostridium septicum alpha toxin","volume":"45","author":"Bentsen","year":"2006","journal-title":"Biochemistry"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"84","DOI":"10.1111\/j.1574-6968.2009.01865.x","article-title":"Identification of amino acids required for receptor binding and toxicity of the Bacillus sphaericus toxin","volume":"303","author":"Singkhamanan","year":"2010","journal-title":"FEMS Microbiol. Lett."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"2778","DOI":"10.1021\/ci200227u","article-title":"LigPlot+: Multiple ligand\u2013protein interaction diagrams for drug discovery","volume":"51","author":"Laskowski","year":"2011","journal-title":"J. Chem. Inf. Model."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"1459","DOI":"10.1021\/acs.biochem.1c00139","article-title":"Comparative perturbation-based modeling of the SARS-CoV-2 spike protein binding with host receptor and neutralizing antibodies: Structurally adaptable allosteric communication hotspots define spike sites targeted by global circulating mutations","volume":"60","author":"Verkhivker","year":"2021","journal-title":"Biochemistry"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"721","DOI":"10.1107\/S1744309110015344","article-title":"Crystallization and preliminary X-ray crystallographic analysis of a full-length active form of the Cry4Ba toxin from Bacillus thuringiensis","volume":"66","author":"Thamwiriyasati","year":"2010","journal-title":"Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"20793","DOI":"10.1074\/jbc.M114.627554","article-title":"Potential prepore trimer formation by the Bacillus thuringiensis mosquito-specific toxin: Molecular insights into a critical prerequisite of membrane-bound monomers","volume":"290","author":"Sriwimol","year":"2015","journal-title":"J. Biol. Chem."}],"container-title":["Toxins"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2072-6651\/15\/2\/114\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T18:18:59Z","timestamp":1760120339000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2072-6651\/15\/2\/114"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,1,29]]},"references-count":40,"journal-issue":{"issue":"2","published-online":{"date-parts":[[2023,2]]}},"alternative-id":["toxins15020114"],"URL":"https:\/\/doi.org\/10.3390\/toxins15020114","relation":{},"ISSN":["2072-6651"],"issn-type":[{"value":"2072-6651","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,1,29]]}}}