We’d love to hear ideas for where phosphorylation - or PTM-specific binders could make a difference. If you’re interested in collaborating, please reach out! (8/8)

This was only possible in a team effort with great people! Thanks @jasonzxzhang.bsky.social, Kejia Wu, Brian Coventry, Gyu Rie Lee, Kody Klupt, Jiuhan Shi, Rafael Brent and the entire @uwproteindesign.bsky.social, with guidance from David Baker and many collaborators. 🙏 (7/8)

Specificity matters. An all-by-all assay showed strong diagonal binding - each binder recognized only its cognate phosphopeptide, with negligible cross-reactivity. (6/8)

Crystal structures of CD3ε and EGFR complexes matched design models within ~2 Å. The intended phosphate-binding motifs were reproduced with atomic accuracy, validating the approach. (5/8)

We tested four sites: CD3ε (TCR), EGFR pY1068 & pY1173, INSR pY1361. In each case, RFD2-MI produced compact proteins that selectively bound the phosphorylated peptide, confirmed by yeast display & BLI with comparable affinities to native phosphorylation binding domains. (4/8)

We built RFD2-MI, an all-atom diffusion model for molecular interfaces based on RFD2. It co-designs binder + peptide and uses 1D conditioning features like hotspots, secondary structure, and solvent exposure to steer phosphate-pocket formation. (3/8)

Existing tools fall short: antibodies can’t always tell sites apart, and natural domains like SH2 struggle with specificity. We needed a way to design de novo binders that are both phosphorylation- and sequence-specific. (2/8)

I would also be very interested in find some alternative letters, for example a B and P for a new ‘I ❤️ Biophysics’ sticker. Currently its hard to read for some people even with the Howarth and Chroma alphabet. SARS-CoV-2 NSP13 provided an amazing heart btw!