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However, the structural and mechanistic basis for protease activity in the S9D subfamily, restricted to photosynthetic eukaryotes (e.g., plants), cyanobacteria, proteobacteria and flavobacteria, is unknown. Here, we report the first high\u2010resolution cryo\u2010EM structures of an S9D protease, chloroplast glutamyl endopeptidase (CGEP) from the model plant\n                    <jats:italic>Arabidopsis thaliana<\/jats:italic>\n                    . CGEP adopts a dimeric architecture stabilized by two distinct interfaces: hydrophobic interactions between catalytic domains and an interdomain \u03b2\u2010sheet linking the cap and catalytic domains. These interactions create a scaffold that supports a hinge loop, which acts as a steric gate to restrict substrate access and confine catalytic activity to the closed conformation. Unlike S9A\u2010B\u2010C proteases, CGEP maintains an intact catalytic triad in both open and closed states, relying on hinge\u2010loop gating rather than catalytic disruption for regulation. Structural analysis and mutagenesis reveal that the hinge loop forms a conserved pocket favoring glutamate side chains, explaining CGEP's strong glutamate preference at cleavage sites. Together, these findings uncover a unique regulatory paradigm for S9D proteases and provide a structural framework for understanding substrate selectivity and dimerization.\n                  <\/jats:p>","DOI":"10.1002\/pro.70624","type":"journal-article","created":{"date-parts":[[2026,5,18]],"date-time":"2026-05-18T05:54:13Z","timestamp":1779083653000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Structural basis for dimerization, catalytic regulation, and substrate selectivity of the chloroplast\n                    <scp>S9D CGEP<\/scp>\n                    protease in\n                    <i>Arabidopsis thaliana<\/i>"],"prefix":"10.1002","volume":"35","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8761-7198","authenticated-orcid":false,"given":"Jacqueline J.","family":"Ehrlich","sequence":"first","affiliation":[{"name":"Department of Biomedical and Translational Sciences Cornell University  Ithaca New York USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pratyush","family":"Routray","sequence":"additional","affiliation":[{"name":"School of Integrative Plant Science, Plant Biology Section Cornell University  Ithaca New York USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0000-8453-7638","authenticated-orcid":false,"given":"Louis","family":"Enns","sequence":"additional","affiliation":[{"name":"Department of Biomedical and Translational Sciences Cornell University  Ithaca New York USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Klaas J.","family":"van Wijk","sequence":"additional","affiliation":[{"name":"School of Integrative Plant Science, Plant Biology Section Cornell University  Ithaca New York USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Toshimitsu","family":"Kawate","sequence":"additional","affiliation":[{"name":"Department of Biomedical and Translational Sciences Cornell University  Ithaca New York USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,5,17]]},"reference":[{"key":"e_1_2_9_2_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-025-62068-3"},{"key":"e_1_2_9_3_1","doi-asserted-by":"publisher","DOI":"10.1104\/pp.20.00752"},{"key":"e_1_2_9_4_1","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkae300"},{"key":"e_1_2_9_5_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.biochem.9b00031"},{"key":"e_1_2_9_6_1","doi-asserted-by":"publisher","DOI":"10.1111\/j.1399-3054.2005.00441.x"},{"key":"e_1_2_9_7_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0092-8674(00)81416-6"},{"key":"e_1_2_9_8_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.xplc.2022.100424"},{"key":"e_1_2_9_9_1","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkr366"},{"key":"e_1_2_9_10_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M110.169862"},{"key":"e_1_2_9_11_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0006-291X(03)00258-4"},{"key":"e_1_2_9_12_1","doi-asserted-by":"publisher","DOI":"10.1038\/s42003-023-04428-7"},{"key":"e_1_2_9_13_1","doi-asserted-by":"publisher","DOI":"10.2174\/1389203720666190925103339"},{"key":"e_1_2_9_14_1","doi-asserted-by":"publisher","DOI":"10.1039\/D2SC02276A"},{"key":"e_1_2_9_15_1","doi-asserted-by":"publisher","DOI":"10.1002\/pro.70320"},{"key":"e_1_2_9_16_1","doi-asserted-by":"publisher","DOI":"10.1104\/pp.114.2.715"},{"key":"e_1_2_9_17_1","doi-asserted-by":"publisher","DOI":"10.1042\/EBC20210058"},{"key":"e_1_2_9_18_1","doi-asserted-by":"publisher","DOI":"10.1105\/tpc.17.00481"},{"key":"e_1_2_9_19_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M113.451534"},{"key":"e_1_2_9_20_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00425-012-1614-1"},{"key":"e_1_2_9_21_1","doi-asserted-by":"publisher","DOI":"10.1128\/JB.02010-07"},{"key":"e_1_2_9_22_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.molp.2016.08.005"},{"key":"e_1_2_9_23_1","doi-asserted-by":"publisher","DOI":"10.1182\/blood-2013-02-487470"},{"key":"e_1_2_9_24_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00018-002-8427-5"},{"key":"e_1_2_9_25_1","doi-asserted-by":"publisher","DOI":"10.1038\/nsb882"},{"key":"e_1_2_9_26_1","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkx1134"},{"key":"e_1_2_9_27_1","doi-asserted-by":"publisher","DOI":"10.1002\/pro.3948"},{"key":"e_1_2_9_28_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jbc.2022.101609"},{"key":"e_1_2_9_29_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.jcim.3c00054"},{"key":"e_1_2_9_30_1","doi-asserted-by":"publisher","DOI":"10.1093\/plcell\/koae178"},{"key":"e_1_2_9_31_1","doi-asserted-by":"publisher","DOI":"10.1107\/S2059798321010810"},{"key":"e_1_2_9_32_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.RA118.004254"},{"key":"e_1_2_9_33_1","doi-asserted-by":"publisher","DOI":"10.1093\/jb\/mvg138"},{"key":"e_1_2_9_34_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M212355200"},{"key":"e_1_2_9_35_1","doi-asserted-by":"publisher","DOI":"10.1002\/jcc.24249"}],"container-title":["Protein 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