{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,10,23]],"date-time":"2026-10-23T21:19:21Z","timestamp":1792790361933,"version":"4.5.0"},"posted":{"date-parts":[[2026,9,23]]},"group-title":"Biochemistry","reference-count":39,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,9,23]],"date-time":"2026-09-23T00:00:00Z","timestamp":1790121600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"funder":[{"name":"National Institute of General Medical Sciences","award":["R35GM128923"],"award-info":[{"award-number":["R35GM128923"]}],"id":[{"id":"https:\/\/ror.org\/04q48ey07","id-type":"ROR","asserted-by":"crossref"}]},{"name":"National Science Foundation","award":["2240095"],"award-info":[{"award-number":["2240095"]}]},{"name":"Welch Foundation","award":["AT-1918\u201320170325"],"award-info":[{"award-number":["AT-1918\u201320170325"]}],"id":[{"id":"https:\/\/ror.org\/00np6vq88","id-type":"ROR","asserted-by":"crossref"}]},{"name":"Welch Foundation","award":["AT-2060-20210327"],"award-info":[{"award-number":["AT-2060-20210327"]}],"id":[{"id":"https:\/\/ror.org\/00np6vq88","id-type":"ROR","asserted-by":"crossref"}]},{"name":"Welch Foundation","award":["AT-2060-20240404"],"award-info":[{"award-number":["AT-2060-20240404"]}],"id":[{"id":"https:\/\/ror.org\/00np6vq88","id-type":"ROR","asserted-by":"crossref"}]},{"name":"National Institute of Allergy and Infectious Diseases","award":["R01AI178692"],"award-info":[{"award-number":["R01AI178692"]}],"id":[{"id":"https:\/\/ror.org\/043z4tv69","id-type":"ROR","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,9,23]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Nitrate is an important oxyanion in bacterial physiology, with major roles as a nitrogen source for assimilation and as an alternative electron acceptor in anaerobic respiration. Accordingly, diverse nitrate-responsive regulatory systems sense nitrate availability and control the expression of genes involved in nitrate transport and metabolism. In the NreABC system, the soluble GAF-like domain protein NreA functions as a cellular nitrate sensor. To date, our molecular understanding of nitrate recognition by NreA has been derived primarily from studies of the\n                  <jats:italic>Staphylococcus carnosus<\/jats:italic>\n                  protein, with less known about homologues from other species. Here, we identified 736 NreA homologues through bioinformatic analysis of the GAF-like domain superfamily and selected five staphylococcal representatives for thermodynamic characterization by isothermal titration calorimetry. Despite conservation of the nitrate-binding pocket, these homologues resolved into high- and low-affinity groups with distinct thermodynamic profiles. Comparative structural modeling and chimeragenesis of a low-affinity homologue revealed that the C-terminal region is a non-coordinating determinant of nitrate-binding affinity. Together, these findings show that a conserved nitrate-binding pocket preserves recognition, while distal sequence variation shapes thermodynamics and tunes the nitrate concentration range over which NreA sensors can function.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>Abstract Figure<\/jats:title>\n                  <jats:fig id=\"ufig1\" position=\"float\" orientation=\"portrait\" fig-type=\"figure\">\n                    <jats:graphic xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" xlink:href=\"751059v1_ufig1\" position=\"float\" orientation=\"portrait\"\/>\n                  <\/jats:fig>\n                <\/jats:sec>","DOI":"10.64898\/2026.09.22.751059","type":"posted-content","created":{"date-parts":[[2026,9,23]],"date-time":"2026-09-23T16:55:11Z","timestamp":1790182511000},"source":"Crossref","is-referenced-by-count":0,"title":["Thermodynamic Diversity in Staphylococcal Nitrate Sensors with a Conserved Binding Pocket"],"prefix":"10.64898","author":[{"given":"Elizabeth K.","family":"Pack","sequence":"first","affiliation":[{"name":"Department of Chemistry and Biochemistry, The University of Texas at Dallas, Richardson, TX 75080"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ke","family":"Ji","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biochemistry, The University of Texas at Dallas, Richardson, TX 75080"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jonathan","family":"Martin","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences, The University of Texas at Dallas, Richardson, TX 75080"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Weicheng","family":"Peng","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biochemistry, The University of Texas at Dallas, Richardson, TX 75080"},{"name":"Department of Biological Sciences, The University of Texas at Dallas, Richardson, TX 75080"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mariah A.","family":"Cook","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biochemistry, The University of Texas at Dallas, Richardson, TX 75080"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gabriele","family":"Meloni","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biochemistry, The University of Texas at Dallas, Richardson, TX 75080"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sheel C.","family":"Dodani","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biochemistry, The University of Texas at Dallas, Richardson, TX 75080"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026092622250907000_2026.09.22.751059v1.1","doi-asserted-by":"publisher","DOI":"10.1038\/S41586-024-07487-W"},{"key":"2026092622250907000_2026.09.22.751059v1.2","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkae1010"},{"key":"2026092622250907000_2026.09.22.751059v1.3","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkae1082"},{"key":"2026092622250907000_2026.09.22.751059v1.4","doi-asserted-by":"crossref","unstructured":"4. 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