{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,9,20]],"date-time":"2025-09-20T20:17:31Z","timestamp":1758399451206,"version":"3.40.5"},"reference-count":44,"publisher":"Wiley","issue":"11","license":[{"start":{"date-parts":[[2020,6,4]],"date-time":"2020-06-04T00:00:00Z","timestamp":1591228800000},"content-version":"am","delay-in-days":349,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#am"},{"start":{"date-parts":[[2019,6,21]],"date-time":"2019-06-21T00:00:00Z","timestamp":1561075200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"funder":[{"DOI":"10.13039\/100000001","name":"National Science Foundation","doi-asserted-by":"publisher","award":["1530862"],"award-info":[{"award-number":["1530862"]}],"id":[{"id":"10.13039\/100000001","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["1R15AI124108\u201001A1"],"award-info":[{"award-number":["1R15AI124108\u201001A1"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Proteins"],"published-print":{"date-parts":[[2019,11]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>Like many Gram\u2010negative pathogens, <jats:italic>Shigella<\/jats:italic> rely on a type three secretion system (T3SS) for injection of effector proteins directly into eukaryotic host cells to initiate and sustain infection. Protein secretion through the needle\u2010like type three secretion apparatus (T3SA) requires ATP hydrolysis by the T3SS ATPase Spa47, making it a likely target for in vivo regulation of T3SS activity and an attractive target for small molecule therapeutics against shigellosis. Here, we developed a model of an activated Spa47 homo\u2010hexamer, identifying two distinct regions at each protomer interface that we hypothesized to provide intermolecular interactions supporting Spa47 oligomerization and enzymatic activation. Mutational analysis and a series of high\u2010resolution crystal structures confirm the importance of these residues, as many of the engineered mutants are unable to form oligomers and efficiently hydrolyze ATP in vitro. Furthermore, in vivo evaluation of <jats:italic>Shigella<\/jats:italic> virulence phenotype uncovered a strong correlation between T3SS effector protein secretion, host cell membrane disruption, and cellular invasion by the tested mutant strains, suggesting that perturbation of the identified interfacial residues\/interactions influences Spa47 activity through preventing oligomer formation, which in turn regulates <jats:italic>Shigella<\/jats:italic> virulence. The most impactful mutations are observed within the conserved Site 2 interface where the native residues support oligomerization and likely contribute to a complex hydrogen bonding network that organizes the active site and supports catalysis. The critical reliance on these conserved residues suggests that aspects of T3SS regulation may also be conserved, providing promise for the development of a cross\u2010species therapeutic that broadly targets T3SS ATPase oligomerization and activation.<\/jats:p>","DOI":"10.1002\/prot.25754","type":"journal-article","created":{"date-parts":[[2019,6,4]],"date-time":"2019-06-04T17:13:34Z","timestamp":1559668414000},"page":"931-942","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["Interfacial amino acids support Spa47 oligomerization and <i>shigella<\/i> type three secretion system activation"],"prefix":"10.1002","volume":"87","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3508-9554","authenticated-orcid":false,"given":"Hannah J.","family":"Demler","sequence":"first","affiliation":[{"name":"Department of Chemistry and Biochemistry Utah State University  Logan 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