Programmed inhibition of an innate immune receptor via de novo designed transmembrane proteins

Transmembrane domains of immune complexes transmit precise signals across lipid bilayers. Probing their interactions has the potential to yield mechanistic insights relevant to therapeutic design. However, our capability to generate molecules directed to bind lipid-embedded sites is limited. Here, we demonstrate a computational strategy to design polypeptides targeting Toll-like receptor 4 (TLR4), a mediator of inflammatory signaling, directly within membranes. TLR4 poses a formidable molecular recognition challenge, as its transmembrane domain is largely apolar, lacks a defined sequence motif, and exhibits an underdetermined structure–function relationship. One synthetic protein binds TLR4’s transmembrane domain and antagonizes NFκB signaling in human cells, proving that precise transmembrane domain interactions are essential for cross-membrane conformational coupling. This work refines design principles for encoding stable interactions in cellular membranes and expands the range of lipid-embedded mechanisms accessible to probe with computationally derived molecules.