Department of Genetics, Stanford University, Stanford, United States
Jagoree Roy
Department of Biology, Stanford University, Stanford, United States
Björn Harink
Department of Genetics, Stanford University, Stanford, United States
Nikhil P Damle
Department of Biology, Stanford University, Stanford, United States
Naomi R Latorraca
Biophysics Program, Stanford University, Stanford, United States
Brian C Baxter
Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States
Kara Brower
Department of Bioengineering, Stanford University, Stanford, United States
Scott A Longwell
Department of Bioengineering, Stanford University, Stanford, United States
Tanja Kortemme
Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, United States; Chan Zuckerberg Biohub, San Francisco, United States
Kurt S Thorn
Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States
Department of Genetics, Stanford University, Stanford, United States; Department of Bioengineering, Stanford University, Stanford, United States; Chan Zuckerberg Biohub, San Francisco, United States; ChEM-H Institute, Stanford University, Stanford, United States
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.
