Quantitative mapping of protein-peptide affinity landscapes using spectrally encoded beads

• DOI: 10.7554/elife.40499
• Publication Date: 2019-07-08T14:00:16Z
• Authors: Huy Quoc Nguyen; Jagoree Roy; Björn Harink; Nikhil P Damle; Naomi R Latorraca; Brian C Baxter; Kara Brower; Scott A Longwell; Tanja Kortemme; Kurt S Thorn; Martha S Cyert; Polly Morrell For...
• Keywords: 0301 basic medicine; QH301-705.5; Science; microfluidics; phophatase specificity; Binding, Competitive; 03 medical and health sciences; Biochemistry and Chemical Biology; Peptide Library; Phosphoprotein Phosphatases; Humans; Amino Acid Sequence; Protein Interaction Maps; Biology (General); calcineurin; Calcineurin; protein specificity; Q; R; Proteins; Hydrogels; Models, Theoretical; Microspheres; protein-peptide interactions; spectral encoding; Medicine; Peptides; Protein Processing, Post-Translational; Algorithms; Protein Binding

Transient, regulated binding of globular protein domains to Short Linear Motifs (SLiMs) in disordered regions of other proteins drives cellular signaling. Mapping the energy landscapes of these interactions is essential for deciphering and perturbing signaling networks but is challenging due to their weak affinities. We present a powerful technology (MRBLE-pep) that simultaneously quantifies protein binding to a library of peptides directly synthesized on beads containing unique spectral codes. Using MRBLE-pep, we systematically probe binding of calcineurin (CN), a conserved protein phosphatase essential for the immune response and target of immunosuppressants, to the PxIxIT SLiM. We discover that flanking residues and post-translational modifications critically contribute to PxIxIT-CN affinity and identify CN-binding peptides based on multiple scaffolds with a wide range of affinities. The quantitative biophysical data provided by this approach will improve computational modeling efforts, elucidate a broad range of weak protein-SLiM interactions, and revolutionize our understanding of signaling networks.