{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T17:09:01Z","timestamp":1785431341221,"version":"3.56.0"},"posted":{"date-parts":[[2026,6,23]]},"group-title":"Biochemistry","reference-count":46,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,6,23]],"date-time":"2026-06-23T00:00:00Z","timestamp":1782172800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"funder":[{"DOI":"10.13039\/100000057","name":"National Institute of General Medical Sciences","doi-asserted-by":"crossref","award":["GM114611"],"award-info":[{"award-number":["GM114611"]}],"id":[{"id":"10.13039\/100000057","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100014989","name":"Chan Zuckerberg Initiative","doi-asserted-by":"crossref","id":[{"id":"10.13039\/100014989","id-type":"DOI","asserted-by":"crossref"}]},{"id":[{"id":"https:\/\/ror.org\/02a7hjv13","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,6,23]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Peptidoglycan biogenesis requires membrane flippases to translocate lipid-linked precursors across the cytoplasmic membrane for processing (1). This essential step is mediated by MurJ, the lipid II flippase conserved across all peptidoglycan-producing bacteria (2). While MurJ from diderm bacteria has been structurally resolved in multiple conformational states (3\u20136), its monoderm homolog remains uncharacterized. Monoderm MurJ homologs exhibit substantial sequence divergence yet retain the same lipid II flipping function (7) and are promising antibiotic targets. Here we report structures of\n                  <jats:italic>Staphylococcus aureus<\/jats:italic>\n                  MurJ (\n                  <jats:italic>Sa<\/jats:italic>\n                  MurJ) captured in both outward- and inward-facing conformations. These structures show that\n                  <jats:italic>Sa<\/jats:italic>\n                  MurJ adopts the conserved MOP family fold and undergoes conformational transitions consistent with an alternating-access mechanism. Our findings reveal conserved and divergent features of MurJ between diderm and monoderm bacteria that are critical for lipid II flipping and provide a structural framework for probing substrate recognition and specific inhibition.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>Significance Statement<\/jats:title>\n                  <jats:p>\n                    The growing global threat of antibiotic resistance and the limited development of new antibacterial therapies underscore the urgent need to identify and mechanistically characterize new antibiotic targets and mechanisms. MurJ is an essential membrane transporter required for cell wall biosynthesis and represents an attractive but unexplored antibiotic target. Here we determine the structures of MurJ from a clinically critical monoderm pathogen\n                    <jats:italic>Staphylococcus aureus<\/jats:italic>\n                    in key conformational states during its transport cycle. This work advances our understanding of an essential step in bacterial cell wall synthesis, reveals key distinctions between monoderm and diderm MurJ, and defines structural features that can be exploited for antibiotic discovery.\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2026.06.21.733627","type":"posted-content","created":{"date-parts":[[2026,6,23]],"date-time":"2026-06-23T11:10:36Z","timestamp":1782213036000},"source":"Crossref","is-referenced-by-count":0,"title":["The structure of the lipid II flippase from monoderm bacteria"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7110-2448","authenticated-orcid":false,"given":"Yancheng E.","family":"Li","sequence":"first","affiliation":[{"name":"Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0008-3396-3117","authenticated-orcid":false,"given":"Grace F.","family":"Baron","sequence":"additional","affiliation":[{"name":"Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0021-889X","authenticated-orcid":false,"suffix":"Jr.","given":"William M.","family":"Clemons","sequence":"additional","affiliation":[{"name":"Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California, USA"},{"name":"Chan Zuckerberg Initiative, Redwood City, California, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026062409301114000_2026.06.21.733627v1.1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.chemrev.1c00773"},{"key":"2026062409301114000_2026.06.21.733627v1.2","doi-asserted-by":"publisher","DOI":"10.1126\/science.1254522"},{"key":"2026062409301114000_2026.06.21.733627v1.3","doi-asserted-by":"crossref","first-page":"171","DOI":"10.1038\/nsmb.3346","article-title":"Crystal structure of the MOP flippase MurJ in an inward-facing conformation","volume":"24","year":"2017","journal-title":"Nature Structural & Molecular Biology"},{"key":"2026062409301114000_2026.06.21.733627v1.4","doi-asserted-by":"crossref","unstructured":"S. Zheng , et al., Structure and mutagenic analysis of the lipid II flippase MurJ from Escherichia coli. 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