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

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Cite this article as: eLife 2019;8:e40499 doi: 10.7554/eLife.40499

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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 human 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.

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Author details

Huy Quoc Nguyen

Department of Genetics, Stanford University, Stanford, United States

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The authors declare that no competing interests exist.
Jagoree Roy

Department of Biology, Stanford University, Stanford, United States

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The authors declare that no competing interests exist.
Bjorn Harink

Department of Genetics, Stanford University, Stanford, United States

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The authors declare that no competing interests exist.
Nikhil P Damle

Department of Biology, Stanford University, Stanford, United States

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The authors declare that no competing interests exist.
Naomi R Latorraca

Biophysics Program, Stanford University, Stanford, United States

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The authors declare that no competing interests exist.
Brian C Baxter

Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States

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The authors declare that no competing interests exist.
Kara Brower

Department of Bioengineering, Stanford University, Stanford, United States

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The authors declare that no competing interests exist.
Scott A Longwell

Department of Bioengineering, Stanford University, Stanford, United States

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Tanja Kortemme

Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, United States

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The authors declare that no competing interests exist.
Kurt S Thorn

Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States

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The authors declare that no competing interests exist.
Martha S Cyert

Department of Biology, Stanford University, Stanford, United States

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The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0003-3825-7437
Polly Morrell Fordyce

Department of Genetics, Stanford University, Stanford, United States

For correspondence
pfordyce@stanford.edu

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The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-9505-0638

Funding

National Institute of General Medical Sciences (DP2GM123641)

Polly Morrell Fordyce

National Institute of General Medical Sciences (R01GM107132)

Kurt S Thorn

National Institute of General Medical Sciences (R01GM119336)

Martha S Cyert

National Institute of General Medical Sciences (R01GM117189)

Tanja Kortemme

National Institute of General Medical Sciences (R01GM110089)

Tanja Kortemme

Chan Zuckerberg Biohub

Tanja Kortemme

Chan Zuckerberg Biohub

Polly Morrell Fordyce

Sloan Foundation

Polly Morrell Fordyce

Beckman Foundation

Polly Morrell Fordyce

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

Michael A Marletta, University of California, Berkeley, United States

Publication history

Received: August 8, 2018
Accepted: July 3, 2019
Accepted Manuscript published: July 8, 2019 (version 1)
Version of Record published: September 5, 2019 (version 2)

Copyright

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.