Quantitative mapping of protein-peptide affinity landscapes using spectrally encoded beads
Open Access
- 8 July 2019
- journal article
- research article
- Published by eLife Sciences Publications, Ltd in eLife
Abstract
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.Keywords
Funding Information
- National Institute of General Medical Sciences (DP2GM123641)
- National Institute of General Medical Sciences (R01GM107132)
- National Institute of General Medical Sciences (R01GM119336)
- National Institute of General Medical Sciences (R01GM117189)
- National Institute of General Medical Sciences (R01GM110089)
- Chan Zuckerberg Biohub
- Alfred P. Sloan Foundation
- Arnold and Mabel Beckman Foundation
This publication has 81 references indexed in Scilit:
- Histone Recognition and Large-Scale Structural Analysis of the Human Bromodomain FamilyCell, 2012
- Intrinsic disorder: signaling via highly specific but short-lived associationTrends in Biochemical Sciences, 2012
- Measuring Binding of Protein to Gel-Bound Ligands Using Magnetic LevitationJournal of the American Chemical Society, 2012
- Recent theoretical and computational advances for modeling protein–ligand binding affinitiesAdvances in Protein Chemistry and Structural Biology, 2011
- High-Throughput Screening of One-Bead-One-Compound Libraries: Identification of Cyclic Peptidyl Inhibitors against Calcineurin/NFAT InteractionACS Combinatorial Science, 2011
- Rosetta3Methods in Enzymology, 2010
- On-Bead Screening of Combinatorial Libraries: Reduction of Nonspecific Binding by Decreasing Surface Ligand DensityJournal of Combinatorial Chemistry, 2009
- New assay to detect low‐affinity interactions and characterization of leukocyte receptors for collagen including leukocyte‐associated Ig‐like receptor‐1 (LAIR‐1)European Journal of Immunology, 2009
- A Conserved Docking Surface on Calcineurin Mediates Interaction with Substrates and ImmunosuppressantsMolecular Cell, 2009
- A Conserved Docking Site Modulates Substrate Affinity for Calcineurin, Signaling Output, and In Vivo FunctionMolecular Cell, 2007