{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,9,11]],"date-time":"2026-09-11T23:14:53Z","timestamp":1789168493359,"version":"build-2803163510"},"posted":{"date-parts":[[2026,9,9]]},"group-title":"Biochemistry","reference-count":36,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,9,9]],"date-time":"2026-09-09T00:00:00Z","timestamp":1788912000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000774","name":"The Defense Threat Reduction Agency","doi-asserted-by":"crossref","award":["HDTRA1-22-1-0012"],"award-info":[{"award-number":["HDTRA1-22-1-0012"]}],"id":[{"id":"10.13039\/100000774","id-type":"DOI","asserted-by":"crossref"}]},{"name":"NIH","award":["R35 GM136218"],"award-info":[{"award-number":["R35 GM136218"]}]},{"name":"National Science and Technology Council Overseas Project for Post Graduate Research","award":["NSTC 114-2917-I-006-004"],"award-info":[{"award-number":["NSTC 114-2917-I-006-004"]}]},{"name":"The Fund for Shanxi 1331 Project","award":["1331KSC"],"award-info":[{"award-number":["1331KSC"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,9,9]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Acyl-homoserine lactone (AHL) quorum sensing enables many species of proteobacteria to coordinate collective behaviors. In such systems, a synthase produces an AHL signal, which is sensed by a LuxR-family receptor. Despite extensive genomic annotation of LuxR homologs, preferred AHLs for most receptors remain unknown, limiting functional understanding of quorum sensing across diverse bacteria. Here, we present the COPAL (combining ordered predictions of audited ligands) pipeline, which integrates multiple protein-ligand co-folding models to identify preferred AHLs for a specific LuxR. Benchmarking on a leakage-controlled subset of 96 experimentally characterized LuxR-AHL pairs shows that COPAL places the preferred AHL within the top-6 candidates (out of 58) for 68% of receptors, outperforming every individual co-folding model. Further, inter-model agreement correlates with ranking accuracy, offering an indication of confidence. We show that COPAL resolves the specificity shift induced by three-point mutations in LasR and correctly nominates C8-HSL as the preferred ligand for the previously uncharacterized\n                  <jats:italic>Mesorhizobium<\/jats:italic>\n                  sp. NJ3 receptor, which we verified experimentally. Finally, we release the Ranked AHL-LuxR Prediction Hub (RALPH), comprising precomputed rankings for about 10,000 unique LuxR homologs. More broadly, COPAL shows that unweighted rank aggregation of complementary co-folding models offers a general strategy for predicting receptor-ligand specificity in data-scarce biological systems.\n                <\/jats:p>","DOI":"10.64898\/2026.09.04.749498","type":"posted-content","created":{"date-parts":[[2026,9,9]],"date-time":"2026-09-09T14:15:12Z","timestamp":1788963312000},"source":"Crossref","is-referenced-by-count":0,"title":["COPAL: An ensemble of protein-ligand co-folding models enriches preferred ligand predictions for the LuxR-family of quorum sensing receptors"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8423-6525","authenticated-orcid":false,"given":"Davi","family":"Nakajima An","sequence":"first","affiliation":[{"name":"Institute for Protein Design, University of Washington, Seattle, WA"},{"name":"Department of Biochemistry, University of Washington, Seattle, WA"},{"name":"Department of Molecular Engineering, University of Washington, Seattle, WA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0581-054X","authenticated-orcid":false,"given":"Amy L.","family":"Schaefer","sequence":"additional","affiliation":[{"name":"Department of Microbiology, University of Washington, Seattle, WA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-5222-6925","authenticated-orcid":false,"given":"Szu-Min","family":"Chang","sequence":"additional","affiliation":[{"name":"Department of Microbiology, University of Washington, Seattle, WA"},{"name":"current address Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan, Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bianxia","family":"Bai","sequence":"additional","affiliation":[{"name":"Department of Microbiology, University of Washington, Seattle, WA"},{"name":"current address Department of Life Sciences, Changzhi University, Shanxi, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0007-7034-7471","authenticated-orcid":false,"given":"Charlotte I.","family":"Mulligan","sequence":"additional","affiliation":[{"name":"Department of Microbiology, University of Washington, Seattle, WA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2554-7371","authenticated-orcid":false,"given":"Yasuhiro","family":"Oda","sequence":"additional","affiliation":[{"name":"Department of Microbiology, University of Washington, Seattle, WA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9474-8041","authenticated-orcid":false,"given":"E. Peter","family":"Greenberg","sequence":"additional","affiliation":[{"name":"Department of Microbiology, University of Washington, Seattle, WA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7524-8938","authenticated-orcid":false,"given":"Frank","family":"DiMaio","sequence":"additional","affiliation":[{"name":"Institute for Protein Design, University of Washington, Seattle, WA"},{"name":"Department of Biochemistry, University of Washington, Seattle, WA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026091115551226000_2026.09.04.749498v1.1","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-023-05905-z"},{"key":"2026091115551226000_2026.09.04.749498v1.2","doi-asserted-by":"publisher","DOI":"10.1128\/jb.176.2.269-275.1994"},{"key":"2026091115551226000_2026.09.04.749498v1.3","doi-asserted-by":"publisher","DOI":"10.1038\/nrmicro.2016.89"},{"key":"2026091115551226000_2026.09.04.749498v1.4","doi-asserted-by":"publisher","DOI":"10.1038\/nature24624"},{"key":"2026091115551226000_2026.09.04.749498v1.5","doi-asserted-by":"publisher","DOI":"10.1038\/nature07088"},{"key":"2026091115551226000_2026.09.04.749498v1.6","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1114125108"},{"key":"2026091115551226000_2026.09.04.749498v1.7","doi-asserted-by":"publisher","DOI":"10.1021\/cr1000817"},{"key":"2026091115551226000_2026.09.04.749498v1.8","doi-asserted-by":"publisher","DOI":"10.1093\/femsre\/fuw014"},{"key":"2026091115551226000_2026.09.04.749498v1.9","first-page":"e00146","article-title":"Quorum sensing signal selectivity and the potential for interspecies cross talk","volume":"10","year":"2019","journal-title":"mBio"},{"key":"2026091115551226000_2026.09.04.749498v1.10","doi-asserted-by":"crossref","first-page":"e0103922","DOI":"10.1128\/msystems.01039-22","article-title":"Cell-cell signaling proteobacterial LuxR solos: A treasure trove of subgroups having different origins, ligands, and ecological roles","volume":"8","year":"2023","journal-title":"mSystems"},{"key":"2026091115551226000_2026.09.04.749498v1.11","doi-asserted-by":"publisher","DOI":"10.1021\/acs.jcim.1c00203"},{"key":"2026091115551226000_2026.09.04.749498v1.12","doi-asserted-by":"publisher","DOI":"10.1021\/acs.jctc.0c01184"},{"key":"2026091115551226000_2026.09.04.749498v1.13","doi-asserted-by":"crossref","first-page":"e69169","DOI":"10.7554\/eLife.69169","article-title":"A covariation analysis reveals elements of selectivity in quorum sensing systems","volume":"10","year":"2021","journal-title":"Elife"},{"key":"2026091115551226000_2026.09.04.749498v1.14","doi-asserted-by":"crossref","unstructured":"Schaefer, A.L. et al. RefAHL: a curated quorum sensing reference linking diverse LuxI-type signal synthases with their acyl-homoserine lactone products. Microbiol Resour Announc, e0040725 (2025).","DOI":"10.1128\/mra.00644-25"},{"key":"2026091115551226000_2026.09.04.749498v1.15","doi-asserted-by":"publisher","DOI":"10.1038\/S41586-024-07487-W"},{"key":"2026091115551226000_2026.09.04.749498v1.16","doi-asserted-by":"crossref","unstructured":"Passaro, S. , et al. Boltz-2: towards accurate and efficient binding affinity prediction. bioRxiv (2025).","DOI":"10.1101\/2025.06.14.659707"},{"key":"2026091115551226000_2026.09.04.749498v1.17","doi-asserted-by":"crossref","unstructured":"Corley, N. , et al. Accelerating biomolecular modeling with AtomWorks and RF3. bioRxiv (2025).","DOI":"10.1101\/2025.08.14.670328"},{"key":"2026091115551226000_2026.09.04.749498v1.18","doi-asserted-by":"crossref","unstructured":"\u0160krinjar, P. , Eberhardt, J. , Tauriello, G. , Schwede, T. & Durairaj, J. Have protein-ligand cofolding methods moved beyond memorisation? bioRxiv, 2025.02.03.636309 (2025).","DOI":"10.1101\/2025.02.03.636309"},{"key":"2026091115551226000_2026.09.04.749498v1.19","doi-asserted-by":"publisher","DOI":"10.1128\/jb.178.10.2897-2901.1996"},{"key":"2026091115551226000_2026.09.04.749498v1.20","doi-asserted-by":"crossref","first-page":"248","DOI":"10.1038\/ismej.2016.88","article-title":"Association mapping reveals novel serpentine adaptation gene clusters in a population of symbiotic Mesorhizobium","volume":"11","year":"2017","journal-title":"ISME J"},{"key":"2026091115551226000_2026.09.04.749498v1.21","first-page":"e01010","article-title":"A Mesorhizobium japonicum quorum sensing circuit that involves three linked genes and an unusual acyl-homoserine lactone signal","volume":"14","year":"2023","journal-title":"mBio"},{"key":"2026091115551226000_2026.09.04.749498v1.22","doi-asserted-by":"crossref","unstructured":"Wallace, M. , Cummings, D.A. , Roberts, A.G. & Puri, A.W . A widespread methylotroph acyl-homoserine lactone synthase produces a new quorum sensing signal that regulates swarming in Methylobacterium fujisawaense . mBio, e0199923 (2023).","DOI":"10.1101\/2022.10.31.514548"},{"key":"2026091115551226000_2026.09.04.749498v1.23","unstructured":"Bastholm, T. Molecular and genetic characterization of bacterial cell-cell signalling. Ph.D. Thesis, Curtain University (2023)."},{"key":"2026091115551226000_2026.09.04.749498v1.24","doi-asserted-by":"publisher","DOI":"10.1128\/AEM.71.8.4487-4496.2005"},{"key":"2026091115551226000_2026.09.04.749498v1.25","doi-asserted-by":"publisher","DOI":"10.1126\/science.1183605"},{"key":"2026091115551226000_2026.09.04.749498v1.26","doi-asserted-by":"crossref","unstructured":"Luo, S. et al. Structural basis for a bacterial Pip system plant effector recognition protein. Proc Natl Acad Sci U S A 118(2021).","DOI":"10.1073\/pnas.2019462118"},{"key":"2026091115551226000_2026.09.04.749498v1.27","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1809611115"},{"key":"2026091115551226000_2026.09.04.749498v1.28","doi-asserted-by":"publisher","DOI":"10.1002\/jms.1275"},{"key":"2026091115551226000_2026.09.04.749498v1.29","doi-asserted-by":"crossref","unstructured":"Xu, S. et al. Benchmarking all-atom biomolecular structure prediction with FoldBench. Nature Communications 17, 442 (2025).","DOI":"10.1038\/s41467-025-67127-3"},{"key":"2026091115551226000_2026.09.04.749498v1.30","doi-asserted-by":"crossref","first-page":"e0294125","DOI":"10.1128\/mbio.02941-25","article-title":"Modulation of the Pseudomonas aeruginosa quorum sensing cascade by MexT-regulated factors","volume":"16","year":"2025","journal-title":"mBio"},{"key":"2026091115551226000_2026.09.04.749498v1.31","doi-asserted-by":"publisher","DOI":"10.1111\/1462-2920.14262"},{"key":"2026091115551226000_2026.09.04.749498v1.32","doi-asserted-by":"crossref","first-page":"e0163469","DOI":"10.1371\/journal.pone.0163469","article-title":"Identification of unanticipated and novel N-acyl L-homoserine lactones (AHLs) using a sensitive non-targeted LC-MS\/MS method","volume":"11","year":"2016","journal-title":"PLoS One"},{"key":"2026091115551226000_2026.09.04.749498v1.33","doi-asserted-by":"publisher","DOI":"10.1021\/acs.jproteome.1c00849"},{"key":"2026091115551226000_2026.09.04.749498v1.34","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkac976"},{"key":"2026091115551226000_2026.09.04.749498v1.35","doi-asserted-by":"publisher","DOI":"10.1038\/nbt.3988"},{"key":"2026091115551226000_2026.09.04.749498v1.36","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/btab184"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.64898\/2026.09.04.749498","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,11]],"date-time":"2026-09-11T22:55:24Z","timestamp":1789167324000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.64898\/2026.09.04.749498"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,9]]},"references-count":36,"URL":"https:\/\/doi.org\/10.64898\/2026.09.04.749498","relation":{},"subject":[],"published":{"date-parts":[[2026,9,9]]},"subtype":"preprint"}}