De novo design of modular peptide-binding proteins by superhelical matching.

Acknowledgements: We thank B. Wicky, A. Ljubetic and I. Lutz for advice on the split luciferase assay for the second-round design screening; C. Xu for help troubleshooting experiments; T. Schlichtharle for discussion; L. Cao for advice on bio-layer interferometry; H. Pyles for advice on circular dichroism and DHR proteins; R. Hegde for the suggestion to target disordered regions of endogenous proteins; and K. Van Wormer and A. Curtis Smith for laboratory support during COVID-19. This work was supported by the Audacious Project at the Institute for Protein Design (D.B., K.W., M.D., D.A.S. and A.B.); the Michelson Found Animals Foundation grant number GM15-S01 (L.S., K.W. and D.B.); the National Institute on Aging grant 5U19AG065156-02 (D.R.H., K.W. and D.B.); the National Institute of General Medical Sciences grant R35GM128777 (D.C.E.); the Howard Hughes Medical Institute (D.B., W.S. and H.B.); the Open Philanthropy Project Improving Protein Design Fund (Y.-T.C., R.R., C.M.C., G.B., D.C.E. and D.B.); the Donald and Jo Anne Petersen Endowment for Accelerating Advancements in Alzheimer’s Disease Research (T.J.B. and D.B.); a donation from AMGEN to the Institute for Protein Design (I.G.); the Medical Research Council (MC_UP_1201/13 to E.D., T.E.M. and T.J.S.); the Human Frontier Science Program (CDA00034/2017-C to E.D.); and a Sir Henry Wellcome Postdoctoral Fellowship (220480/Z/20/Z to K.E.M.).General approaches for designing sequence-specific peptide-binding proteins would have wide utility in proteomics and synthetic biology. However, designing peptide-binding proteins is challenging, as most peptides do not have defined structures in isolation, and hydrogen bonds must be made to the buried polar groups in the peptide backbone1-3. Here, inspired by natural and re-engineered protein-peptide systems4-11, we set out to design proteins made out of repeating units that bind peptides with repeating sequences, with a one-to-one correspondence between the repeat units of the protein and those of the peptide. We use geometric hashing to identify protein backbones and peptide-docking arrangements that are compatible with bidentate hydrogen bonds between the side chains of the protein and the peptide backbone12. The remainder of the protein sequence is then optimized for folding and peptide binding. We design repeat proteins to bind to six different tripeptide-repeat sequences in polyproline II conformations. The proteins are hyperstable and bind to four to six tandem repeats of their tripeptide targets with nanomolar to picomolar affinities in vitro and in living cells. Crystal structures reveal repeating interactions between protein and peptide interactions as designed, including ladders of hydrogen bonds from protein side chains to peptide backbones. By redesigning the binding interfaces of individual repeat units, specificity can be achieved for non-repeating peptide sequences and for disordered regions of native proteins