{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,14]],"date-time":"2026-10-14T08:49:54Z","timestamp":1791967794898,"version":"4.3.4"},"reference-count":31,"publisher":"Oxford University Press (OUP)","issue":"19","license":[{"start":{"date-parts":[[2026,10,14]],"date-time":"2026-10-14T00:00:00Z","timestamp":1791936000000},"content-version":"vor","delay-in-days":1,"URL":"https:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["32570711"],"award-info":[{"award-number":["32570711"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["32571447"],"award-info":[{"award-number":["32571447"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100005234","name":"CCNU","doi-asserted-by":"publisher","award":["XJ2026004502"],"award-info":[{"award-number":["XJ2026004502"]}],"id":[{"id":"10.13039\/501100005234","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,10,13]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>The methylation-associated defense system (MADS) is a bacterial antiphage immune system that cooperates with CRISPR\u2013Cas systems to restrict phage infection. Within MADS, the MAD3\u2013MAD4 module has been proposed to function as an ATPase\u2013nuclease module related to the OLD nuclease family, but the molecular basis of its assembly and function remains unknown. Here, we combined cryo-electron microscopy, X-ray crystallography, biochemical assays, and mutagenesis to elucidate the architecture and assembly mechanism of the MAD3\u2013MAD4 complex. We found that MAD3 and MAD4 assemble into a stable 2:2 heterotetramer. Structural and biochemical analyses revealed that MAD3 alone lacks detectable ATPase activity, whereas MAD4 is insoluble when expressed alone. Assembly of the MAD3\u2013MAD4 complex induces a conformational rearrangement in which MAD3 adopts an ATPase-competent dimeric conformation, while MAD4 is stabilized through intersubunit interactions. This heterotetrameric assembly exhibits both ATPase and DNA cleavage activities. Overall, our findings reveal the molecular basis of MAD3\u2013MAD4 heterotetramer assembly and provide the first structural and biochemical insights into the function of the MAD3\u2013MAD4 module within the MADS defense system.<\/jats:p>","DOI":"10.1093\/nar\/gkag1001","type":"journal-article","created":{"date-parts":[[2026,10,4]],"date-time":"2026-10-04T11:28:05Z","timestamp":1791113285000},"source":"Crossref","is-referenced-by-count":0,"title":["Structural basis of assembly-dependent activation of the MAD3\u2013MAD4 nuclease in the\n                    <i>Pseudomonas aeruginosa<\/i>\n                    MADS defense system"],"prefix":"10.1093","volume":"54","author":[{"given":"Jin","family":"Zhang","sequence":"first","affiliation":[{"name":"Key Laboratory of Pesticide & Chemical Biology, Ministry of Education, Hubei Key Laboratory of Genetic Regulation and Integrative Biology, School of Life Sciences, Central China Normal University , Wuhan 430079 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zengru","family":"Li","sequence":"additional","affiliation":[{"name":"Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology , Wuhan 430074 ,","place":["China"]},{"name":"Center for Biological Cryo-EM, Huazhong University of Science and Technology , Wuhan 430075 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Le","family":"Ye","sequence":"additional","affiliation":[{"name":"Key Laboratory of Pesticide & Chemical Biology, Ministry of Education, Hubei Key Laboratory of Genetic Regulation and Integrative Biology, School of Life Sciences, Central China Normal University , Wuhan 430079 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yaoyao","family":"Hou","sequence":"additional","affiliation":[{"name":"Key Laboratory of Pesticide & Chemical Biology, Ministry of Education, Hubei Key Laboratory of Genetic Regulation and Integrative Biology, School of Life Sciences, Central China Normal University , Wuhan 430079 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7082-262X","authenticated-orcid":false,"given":"Ke","family":"Liu","sequence":"additional","affiliation":[{"name":"Key Laboratory of Pesticide & Chemical Biology, Ministry of Education, Hubei Key Laboratory of Genetic Regulation and Integrative Biology, School of Life Sciences, Central China Normal University , Wuhan 430079 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5210-3130","authenticated-orcid":false,"given":"Jinrong","family":"Min","sequence":"additional","affiliation":[{"name":"Key Laboratory of Pesticide & Chemical Biology, Ministry of Education, Hubei Key Laboratory of Genetic Regulation and Integrative Biology, School of Life Sciences, Central China Normal University , Wuhan 430079 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2026,10,14]]},"reference":[{"key":"2026101404155629400_B1","doi-asserted-by":"publisher","first-page":"169","DOI":"10.15252\/embj.201489455","article-title":"BREX is a novel phage resistance system widespread in microbial genomes","volume":"34","author":"Goldfarb","year":"2015","journal-title":"EMBO J"},{"key":"2026101404155629400_B2","doi-asserted-by":"publisher","first-page":"522","DOI":"10.1038\/s41586-022-04716-y","article-title":"Phage anti-CBASS and anti-pycsar nucleases subvert bacterial immunity","volume":"605","author":"Hobbs","year":"2022","journal-title":"Nature"},{"key":"2026101404155629400_B3","doi-asserted-by":"publisher","first-page":"691","DOI":"10.1038\/s41586-019-1605-5","article-title":"Cyclic GMP-AMP signalling protects bacteria against viral 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