Structure-guided design of a peptide lock for modular peptide binders.

Peptides play an important role in intra- and intercellular interactions and are frequent analytes in diagnostic assays, also in the form of unstructured, linear epitopes in whole proteins. Yet, due to the many different sequence possibili-ties even for short peptides, classical selection of binding proteins from a library, one at a time, cannot meet the de-mand for new binders. However, modular peptide binders consisting of preselected peptide-binding modules that bind to smaller sequence parts would split the problem into smaller parts. These could then be reassembled to bind to, in principle, arbitrary sequences. Designed Armadillo repeat proteins (dArmRPs) are modular and they do bind elongated peptides in a modular way. Their consensus sequence carries pockets that prefer arginine and lysine, re-spectively. In our quest to select pockets for all amino acid side chains, we had discovered that these repetitive se-quences can lead to register shifts and peptide flipping during selections from libraries, hindering the selection of new binding specificities. To solve this problem, we now created an orthogonal binding specificity by a combination of grafting from beta-catenin and computational design and mutual optimization of the pocket and the bound pep-tide. We have confirmed the design and the desired interactions by x-ray structure determination. Furthermore, we could confirm the absence of sliding in solution by single-molecule Förster resonance energy transfer. The new pocket could be moved from the N-terminus of the protein to the middle, retaining its properties, further underlining the modularity of the system.