1. Structural Biology and Molecular Biophysics

An expanded allosteric network in PTP1B by multitemperature crystallography, fragment screening, and covalent tethering

  1. Daniel A Keedy
  2. Zachary B Hill
  3. Justin T Biel
  4. Emily Kang
  5. T Justin Rettenmaier
  6. Jose Brandao-Neto
  7. Nicholas M Pearce
  8. Frank von Delft
  9. James A Wells
  10. James S Fraser  Is a corresponding author
  1. University of California, San Francisco, United States
  2. Diamond Light Source, United Kingdom
  3. University of Utrecht, Netherlands
https://doi.org/10.7554/eLife.36307
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Cite this article
  1. Daniel A Keedy
  2. Zachary B Hill
  3. Justin T Biel
  4. Emily Kang
  5. T Justin Rettenmaier
  6. Jose Brandao-Neto
  7. Nicholas M Pearce
  8. Frank von Delft
  9. James A Wells
  10. James S Fraser
(2018)
An expanded allosteric network in PTP1B by multitemperature crystallography, fragment screening, and covalent tethering
eLife 7:e36307.
https://doi.org/10.7554/eLife.36307
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Abstract

Allostery is an inherent feature of proteins, but it remains challenging to reveal the mechanisms by which allosteric signals propagate. A clearer understanding of this intrinsic circuitry would afford new opportunities to modulate protein function. Here we have identified allosteric sites in protein tyrosine phosphatase 1B (PTP1B) by combining multiple-temperature X-ray crystallography experiments and structure determination from hundreds of individual small-molecule fragment soaks. New modeling approaches reveal 'hidden' low-occupancy conformational states for protein and ligands. Our results converge on allosteric sites that are conformationally coupled to the active-site WPD loop and are hotspots for fragment binding. Targeting one of these sites with covalently tethered molecules or mutations allosterically inhibits enzyme activity. Overall, this work demonstrates how the ensemble nature of macromolecular structure, revealed here by multitemperature crystallography, can elucidate allosteric mechanisms and open new doors for long-range control of protein function.

Data availability

Data have been deposited in PDB under the accession codes: 6B90, 6B8E, 6B8T, 6B8X, 6B8Z, 6BAI, 6B95, 5QDE, 5QDF, 5QDG, 5QDH, 5QDI, 5QDJ, 5QDK, 5QDL, 5QDM, 5QDN, 5QDO, 5QDP, 5QDQ, 5QDR, 5QDS, 5QDT, 5QDU, 5QDV, 5QDW, 5QDX, 5QDY, 5QDZ, 5QE0, 5QE1, 5QE2, 5QE3, 5QE4, 5QE5, 5QE6, 5QE7, 5QE8, 5QE9, 5QEA, 5QEB, 5QEC, 5QED, 5QEE, 5QEF, 5QEG, 5QEH, 5QEI, 5QEJ, 5QEK, 5QEL, 5QEM, 5QEN, 5QEO, 5QEP, 5QEQ, 5QER, 5QES, 5QET, 5QEU, 5QEV, 5QEW, 5QEX, 5QEY, 5QEZ, 5QF0, 5QF1, 5QF2, 5QF3, 5QF4, 5QF5, 5QF6, 5QF7, 5QF8, 5QF9, 5QFA, 5QFB, 5QFC, 5QFD, 5QFE, 5QFF, 5QFG, 5QFH, 5QFI, 5QFJ, 5QFK, 5QFL, 5QFM, 5QFN, 5QFO, 5QFP, 5QFQ, 5QFR, 5QFS, 5QFT, 5QFU, 5QFV, 5QFW, 5QFX, 5QFY, 5QFZ, 5QG0, 5QG1, 5QG2, 5QG3, 5QG4, 5QG5, 5QG6, 5QG7, 5QG8, 5QG9, 5QGA, 5QGB, 5QGC, 5QGD, 5QGE, 5QGF and further data available at https://zenodo.org/record/1044103

The following data sets were generated

Article and author information

Author details

  1. Daniel A Keedy

    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.

  2. Zachary B Hill

    Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.

  3. Justin T Biel

    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.

  4. Emily Kang

    Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.

  5. T Justin Rettenmaier

    Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.

  6. Jose Brandao-Neto

    XChem, Diamond Light Source, Didcot, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6015-320X

  7. Nicholas M Pearce

    Crystal and Structural Chemistry, University of Utrecht, Utrecht, Netherlands
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6693-8603

  8. Frank von Delft

    XChem, Diamond Light Source, Didcot, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.

  9. James A Wells

    Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8267-5519

  10. James S Fraser

    Department of Bioengineering and Therapeutic Science, California Institute for Quantitative Biology, University of California, San Francisco, San Francisco, United States
    For correspondence
    jfraser@fraserlab.com
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5080-2859

Funding

Kinship Foundation

  • James S Fraser

National Cancer Institute (CA191018)

  • James A Wells

National Cancer Institute ((F31 CA180378)

  • T Justin Rettenmaier

National Institute of General Medical Sciences (GM123159)

  • James S Fraser

National Institute of General Medical Sciences (GM124169)

  • James S Fraser

National Institute of General Medical Sciences (GM124149)

  • James S Fraser

Pew Charitable Trusts

  • James S Fraser

David and Lucile Packard Foundation

  • James S Fraser

National Institute of General Medical Sciences (GM110580)

  • James S Fraser

National Science Foundation (STC-1231306)

  • James S Fraser

University of California (LFR-17-476732)

  • James S Fraser

Helen Hay Whitney Foundation

  • Zachary B Hill

National Cancer Institute (K99CA203002)

  • Zachary B Hill

A.P. Giannini Foundation (Postdoctoral Fellowship)

  • Daniel A Keedy

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

Copyright

© 2018, Keedy 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.

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  1. Daniel A Keedy
  2. Zachary B Hill
  3. Justin T Biel
  4. Emily Kang
  5. T Justin Rettenmaier
  6. Jose Brandao-Neto
  7. Nicholas M Pearce
  8. Frank von Delft
  9. James A Wells
  10. James S Fraser
(2018)
An expanded allosteric network in PTP1B by multitemperature crystallography, fragment screening, and covalent tethering
eLife 7:e36307.
https://doi.org/10.7554/eLife.36307

Share this article

https://doi.org/10.7554/eLife.36307

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    Much of our current understanding of how small-molecule ligands interact with proteins stems from X-ray crystal structures determined at cryogenic (cryo) temperature. For proteins alone, room-temperature (RT) crystallography can reveal previously hidden, biologically relevant alternate conformations. However, less is understood about how RT crystallography may impact the conformational landscapes of protein-ligand complexes. Previously, we showed that small-molecule fragments cluster in putative allosteric sites using a cryo crystallographic screen of the therapeutic target PTP1B (Keedy et al., 2018). Here, we have performed two RT crystallographic screens of PTP1B using many of the same fragments, representing the largest RT crystallographic screens of a diverse library of ligands to date, and enabling a direct interrogation of the effect of data collection temperature on protein-ligand interactions. We show that at RT, fewer ligands bind, and often more weakly – but with a variety of temperature-dependent differences, including unique binding poses, changes in solvation, new binding sites, and distinct protein allosteric conformational responses. Overall, this work suggests that the vast body of existing cryo-temperature protein-ligand structures may provide an incomplete picture, and highlights the potential of RT crystallography to help complete this picture by revealing distinct conformational modes of protein-ligand systems. Our results may inspire future use of RT crystallography to interrogate the roles of protein-ligand conformational ensembles in biological function.

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