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Here, we created synthetic polypeptides targeting a prominent mediator of inflammatory signaling directly within membranes, Toll-like receptor 4 (TLR4), and clarify structural principles of its cross-membrane signaling mechanism. Doing so, we test emerging design principles for computationally encoding protein interactions within lipid and validate a protein\u2013protein interaction screening platform for rapid discovery of de novo transmembrane (TM) protein antagonist of inflammatory signaling via this innate immune receptor. Binding the TM domain of TLR4 poses a formidable de novo molecular recognition challenge not yet achieved, given the membrane-spanning region is predominantly apolar and lacks any recognizable interaction motifs (e.g., sticky small residue repeats). Likewise, TLR4\u2019s structure\u2013function relationship and key residues for oligomerization and cross-membrane signaling are underdetermined. Our engineering strategy identifies lead synthetic TM proteins that specifically bind TLR4\u2019s transmembrane domain and antagonizes NF\u03baB signaling in human cells. These transmembrane-directed chemical probes prove that receptor TM domain interaction geometries are essential for TLR4\u2019s cross-membrane conformational coupling, informing a revised structural mechanism to leverage in future drug design of this protein family. This work refines principles for encoding stable interactions in cellular membranes and expands the range of lipid-embedded mechanisms accessible to probe with computationally derived molecules.<\/jats:p>","DOI":"10.1073\/pnas.2536559123","type":"journal-article","created":{"date-parts":[[2026,9,22]],"date-time":"2026-09-22T16:12:31Z","timestamp":1790093551000},"update-policy":"https:\/\/doi.org\/10.1073\/pnas.cm10313","source":"Crossref","is-referenced-by-count":0,"title":["Programmed inhibition of an innate immune receptor via de novo designed transmembrane proteins"],"prefix":"10.1073","volume":"123","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7050-4464","authenticated-orcid":false,"given":"Colleen A.","family":"Maillie","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02dxx6824","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Integrative Structural and Computational Biology, The Scripps Research Institute","place":["La 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