1. Biochemistry and Chemical Biology
  2. Structural Biology and Molecular Biophysics

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

  1. Huy Quoc Nguyen
  2. Jagoree Roy
  3. Bjorn Harink
  4. Nikhil P Damle
  5. Naomi R Latorraca
  6. Brian C Baxter
  7. Kara Brower
  8. Scott A Longwell
  9. Tanja Kortemme
  10. Kurt S Thorn
  11. Martha S Cyert
  12. Polly Morrell Fordyce  Is a corresponding author
  1. Stanford University, United States
  2. University of California, San Francisco, United States
https://doi.org/10.7554/eLife.40499
Share this article
Cite this article
  1. Huy Quoc Nguyen
  2. Jagoree Roy
  3. Bjorn Harink
  4. Nikhil P Damle
  5. Naomi R Latorraca
  6. Brian C Baxter
  7. Kara Brower
  8. Scott A Longwell
  9. Tanja Kortemme
  10. Kurt S Thorn
  11. Martha S Cyert
  12. Polly Morrell Fordyce
(2019)
Quantitative mapping of protein-peptide affinity landscapes using spectrally encoded beads
eLife 8:e40499.
https://doi.org/10.7554/eLife.40499
  2. Download BibTeX
  3. Download .RIS

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.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files. In addition, all data generated or analyzed during this study are available in an associated public OSF repository (DOI 10.17605/OSF.IO/FPVE2).

The following data sets were generated

Article and author information

Author details

  1. Huy Quoc Nguyen

    Department of Genetics, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.

  2. Jagoree Roy

    Department of Biology, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.

  3. Bjorn Harink

    Department of Genetics, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.

  4. Nikhil P Damle

    Department of Biology, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.

  5. Naomi R Latorraca

    Biophysics Program, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.

  6. Brian C Baxter

    Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.

  7. Kara Brower

    Department of Bioengineering, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.

  8. Scott A Longwell

    Department of Bioengineering, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.

  9. Tanja Kortemme

    Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.

  10. Kurt S Thorn

    Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.

  11. Martha S Cyert

    Department of Biology, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3825-7437

  12. Polly Morrell Fordyce

    Department of Genetics, Stanford University, Stanford, United States
    For correspondence
    pfordyce@stanford.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "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.

Copyright

© 2019, Nguyen et al.

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.

Metrics

  • 3,865
    views
  • 571
    downloads
  • 55
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Huy Quoc Nguyen
  2. Jagoree Roy
  3. Bjorn Harink
  4. Nikhil P Damle
  5. Naomi R Latorraca
  6. Brian C Baxter
  7. Kara Brower
  8. Scott A Longwell
  9. Tanja Kortemme
  10. Kurt S Thorn
  11. Martha S Cyert
  12. Polly Morrell Fordyce
(2019)
Quantitative mapping of protein-peptide affinity landscapes using spectrally encoded beads
eLife 8:e40499.
https://doi.org/10.7554/eLife.40499

Share this article

https://doi.org/10.7554/eLife.40499

Categories and tags

Research organism

Further reading

    1. Biochemistry and Chemical Biology
    2. Genetics and Genomics

    Yerba mate (Ilex paraguariensis) genome provides new insights into convergent evolution of caffeine biosynthesis

    Federico A Vignale, Andrea Hernandez Garcia ... Adrian G Turjanski
    Research Article

    Yerba mate (YM, Ilex paraguariensis) is an economically important crop marketed for the elaboration of mate, the third-most widely consumed caffeine-containing infusion worldwide. Here, we report the first genome assembly of this species, which has a total length of 1.06 Gb and contains 53,390 protein-coding genes. Comparative analyses revealed that the large YM genome size is partly due to a whole-genome duplication (Ip-α) during the early evolutionary history of Ilex, in addition to the hexaploidization event (γ) shared by core eudicots. Characterization of the genome allowed us to clone the genes encoding methyltransferase enzymes that catalyse multiple reactions required for caffeine production. To our surprise, this species has converged upon a different biochemical pathway compared to that of coffee and tea. In order to gain insight into the structural basis for the convergent enzyme activities, we obtained a crystal structure for the terminal enzyme in the pathway that forms caffeine. The structure reveals that convergent solutions have evolved for substrate positioning because different amino acid residues facilitate a different substrate orientation such that efficient methylation occurs in the independently evolved enzymes in YM and coffee. While our results show phylogenomic constraint limits the genes coopted for convergence of caffeine biosynthesis, the X-ray diffraction data suggest structural constraints are minimal for the convergent evolution of individual reactions.

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics

    Recognition and cleavage of human tRNA methyltransferase TRMT1 by the SARS-CoV-2 main protease

    Angel D'Oliviera, Xuhang Dai ... Jeffrey S Mugridge
    Research Article

    The SARS-CoV-2 main protease (Mpro or Nsp5) is critical for production of viral proteins during infection and, like many viral proteases, also targets host proteins to subvert their cellular functions. Here, we show that the human tRNA methyltransferase TRMT1 is recognized and cleaved by SARS-CoV-2 Mpro. TRMT1 installs the N2,N2-dimethylguanosine (m2,2G) modification on mammalian tRNAs, which promotes cellular protein synthesis and redox homeostasis. We find that Mpro can cleave endogenous TRMT1 in human cell lysate, resulting in removal of the TRMT1 zinc finger domain. Evolutionary analysis shows the TRMT1 cleavage site is highly conserved in mammals, except in Muroidea, where TRMT1 is likely resistant to cleavage. TRMT1 proteolysis results in reduced tRNA binding and elimination of tRNA methyltransferase activity. We also determined the structure of an Mpro-TRMT1 peptide complex that shows how TRMT1 engages the Mpro active site in an uncommon substrate binding conformation. Finally, enzymology and molecular dynamics simulations indicate that kinetic discrimination occurs during a later step of Mpro-mediated proteolysis following substrate binding. Together, these data provide new insights into substrate recognition by SARS-CoV-2 Mpro that could help guide future antiviral therapeutic development and show how proteolysis of TRMT1 during SARS-CoV-2 infection impairs both TRMT1 tRNA binding and tRNA modification activity to disrupt host translation and potentially impact COVID-19 pathogenesis or phenotypes.

    1. Biochemistry and Chemical Biology
    2. Microbiology and Infectious Disease

    The nanoscale organization of the Nipah virus fusion protein informs new membrane fusion mechanisms

    Qian Wang, Jinxin Liu ... Qian Liu
    Research Article

    Paramyxovirus membrane fusion requires an attachment protein for receptor binding and a fusion protein for membrane fusion triggering. Nipah virus (NiV) attachment protein (G) binds to ephrinB2 or -B3 receptors, and fusion protein (F) mediates membrane fusion. NiV-F is a class I fusion protein and is activated by endosomal cleavage. The crystal structure of a soluble GCN4-decorated NiV-F shows a hexamer-of-trimer assembly. Here, we used single-molecule localization microscopy to quantify the NiV-F distribution and organization on cell and virus-like particle membranes at a nanometer precision. We found that NiV-F on biological membranes forms distinctive clusters that are independent of endosomal cleavage or expression levels. The sequestration of NiV-F into dense clusters favors membrane fusion triggering. The nano-distribution and organization of NiV-F are susceptible to mutations at the hexamer-of-trimer interface, and the putative oligomerization motif on the transmembrane domain. We also show that NiV-F nanoclusters are maintained by NiV-F–AP-2 interactions and the clathrin coat assembly. We propose that the organization of NiV-F into nanoclusters facilitates membrane fusion triggering by a mixed population of NiV-F molecules with varied degrees of cleavage and opportunities for interacting with the NiV-G/receptor complex. These observations provide insights into the in situ organization and activation mechanisms of the NiV fusion machinery.