{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T14:17:39Z","timestamp":1785421059104,"version":"3.56.0"},"posted":{"date-parts":[[2025,9,26]]},"group-title":"Biochemistry","reference-count":0,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,9,26]],"date-time":"2025-09-26T00:00:00Z","timestamp":1758844800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"}],"funder":[{"award":["R01-DK101451"],"award-info":[{"award-number":["R01-DK101451"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["P20-GM113132"],"award-info":[{"award-number":["P20-GM113132"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["P30-DK117469"],"award-info":[{"award-number":["P30-DK117469"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["P30-GM106394"],"award-info":[{"award-number":["P30-GM106394"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["T32-GM008704"],"award-info":[{"award-number":["T32-GM008704"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["P20-GM103449"],"award-info":[{"award-number":["P20-GM103449"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"name":"Cystic Fibrosis Foundation","award":["STANTO19R0"],"award-info":[{"award-number":["STANTO19R0"]}]},{"name":"Mukovizidose e.V.","award":["S05\/08"],"award-info":[{"award-number":["S05\/08"]}]},{"name":"German Cystic Fibrosis Association"},{"name":"United States Department of Energy","award":["DE-AC02-98CH10886"],"award-info":[{"award-number":["DE-AC02-98CH10886"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,9,26]]},"abstract":"<jats:p>PDZ interaction networks are finely-tuned products of evolution. These widespread binding domains recognize short linear motifs (SLiMs), usually at the C-terminus of their interacting partners, and are involved in trafficking and signaling pathways, the formation of tight junctions, and scaffolding of the post-synaptic density of neurons, amongst other roles. Typically, a single PDZ domain binds multiple targets; conversely, each PDZ-binding protein engages several PDZ domains, dependent on cellular conditions. Historical PDZ binding motifs rely on two key positions for binding. However, previous insights on modulator, or non-motif, selectivity preferences reveal that these limited motifs are insufficient to describe PDZ-mediated interactomes, consistent with the observation that the degree of promiscuity is much more limited than predicted by defined binding classes. Here, we use these principles to engineer and test a peptide-based inhibitor capable of interacting with a single PDZ domain-containing protein in a disease relevant cellular system. We first interrogate a previously developed sequence selective for cystic fibrosis transmembrane conductance regulator (CFTR)-Associated Ligand (CAL), one of five PDZ domains known to bind the CFTR C-terminus, probing for off-target PDZ partners. Once identified, we use parallel biochemical and structural refinement to eliminate these interactions and introduce a CAL PDZ inhibitor with unprecedented PDZ domain selectivity. We test and verify specificity using relevant cellular PDZ target networks in a mass spectrometry-based approach. Our resultant selective inhibitor enhances chloride efflux when applied to polarized patient bronchial epithelial cells, as well as confirms that engineering an effectively single-PDZ peptide is possible when modulator preferences are applied.<\/jats:p>","DOI":"10.1101\/2025.09.25.678608","type":"posted-content","created":{"date-parts":[[2025,9,27]],"date-time":"2025-09-27T04:55:16Z","timestamp":1758948916000},"source":"Crossref","is-referenced-by-count":0,"title":["Sequence engineering at non-motif modulator residues yields a peptide that effectively targets a single PDZ protein in a disease-relevant cellular context"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3646-7521","authenticated-orcid":false,"given":"Jeanine F","family":"Amacher","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Patrick R","family":"Cushing","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lars","family":"Vouilleme","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6477-0459","authenticated-orcid":false,"given":"Sierra N","family":"Cullati","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bin","family":"Deng","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2964-5051","authenticated-orcid":false,"given":"Scott A.","family":"Gerber","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6955-1340","authenticated-orcid":false,"given":"Prisca A.","family":"Boisguerin","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1810-6984","authenticated-orcid":false,"given":"Dean R","family":"Madden","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.1101\/2025.09.25.678608","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,6,24]],"date-time":"2026-06-24T04:58:41Z","timestamp":1782277121000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.1101\/2025.09.25.678608"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,9,26]]},"references-count":0,"URL":"https:\/\/doi.org\/10.1101\/2025.09.25.678608","relation":{"is-preprint-of":[{"id-type":"doi","id":"10.1016\/j.jmb.2025.169597","asserted-by":"subject"}]},"subject":[],"published":{"date-parts":[[2025,9,26]]},"subtype":"preprint"}}