1. Structural Biology and Molecular Biophysics
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The acidic domain of the endothelial membrane protein GPIHBP1 stabilizes lipoprotein lipase activity by preventing unfolding of its catalytic domain

  1. Simon Mysling
  2. Kristian Kølby Kristensen
  3. Mikael Larsson
  4. Anne P Beigneux
  5. Henrik Gårdsvoll
  6. Fong G Loren
  7. André Bensadouen
  8. Thomas JD Jørgensen
  9. Stephen G Young
  10. Michael Ploug  Is a corresponding author
  1. Rigshospitalet, Denmark
  2. University of California, Los Angeles, United States
  3. Rigshospitalet, United States
  4. Cornell University, United States
  5. University of Southern Denmark, Denmark
Research Article
  • Cited 49
  • Views 1,986
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Cite this article as: eLife 2016;5:e12095 doi: 10.7554/eLife.12095

Abstract

GPIHBP1 is a glycolipid-anchored membrane protein of capillary endothelial cells that binds lipoprotein lipase (LPL) within the interstitial space and shuttles it to the capillary lumen. The LPL•GPIHBP1 complex is responsible for margination of triglyceride-rich lipoproteins along capillaries and their lipolytic processing. The current work conceptualizes a model for the GPIHBP1•LPL interaction based on biophysical measurements with hydrogen-deuterium exchange/mass spectrometry, surface plasmon resonance, and zero-length cross-linking. According to this model, GPIHBP1 is comprised of two functionally distinct domains: (1) an intrinsically disordered acidic N-terminal domain; and (2) a folded C-terminal domain that tethers GPIHBP1 to the cell membrane by glycosylphosphatidylinositol. We demonstrate that these domains serve different roles in regulating the kinetics of LPL binding. Importantly, the acidic domain stabilizes LPL catalytic activity by mitigating the global unfolding of LPL's catalytic domain. This study provides a conceptual framework for understanding intravascular lipolysis and GPIHBP1 and LPL mutations causing familial chylomicronemia.

Article and author information

Author details

  1. Simon Mysling

    Finsen Laboratory, Rigshospitalet, Copenhagen, Denmark
    Competing interests
    No competing interests declared.
  2. Kristian Kølby Kristensen

    Finsen Laboratory, Rigshospitalet, Copenhagen, Denmark
    Competing interests
    No competing interests declared.
  3. Mikael Larsson

    Department of Medicine, University of California, Los Angeles, Los Angeles, United States
    Competing interests
    No competing interests declared.
  4. Anne P Beigneux

    Department of Medicine, University of California, Los Angeles, Los Angeles, United States
    Competing interests
    No competing interests declared.
  5. Henrik Gårdsvoll

    Finsen Laboratory, Rigshospitalet, Copenhagen, United States
    Competing interests
    No competing interests declared.
  6. Fong G Loren

    Department of Medicine, University of California, Los Angeles, Loa Angeles, United States
    Competing interests
    No competing interests declared.
  7. André Bensadouen

    Division of Nutritional Science, Cornell University, Ithaca, United States
    Competing interests
    No competing interests declared.
  8. Thomas JD Jørgensen

    Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark
    Competing interests
    No competing interests declared.
  9. Stephen G Young

    Department of Medicine, University of California, Los Angeles, Los Angeles, United States
    Competing interests
    Stephen G Young, Reviewing editor, eLife.
  10. Michael Ploug

    Finsen Laboratory, Rigshospitalet, Copenhagen, Denmark
    For correspondence
    m-ploug@finsenlab.dk
    Competing interests
    No competing interests declared.

Reviewing Editor

  1. Christopher K. Glass, University of California, San Diego, United States

Publication history

  1. Received: October 5, 2015
  2. Accepted: January 2, 2016
  3. Accepted Manuscript published: January 3, 2016 (version 1)
  4. Version of Record published: February 3, 2016 (version 2)
  5. Version of Record updated: May 9, 2016 (version 3)

Copyright

© 2016, Mysling 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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    The Mre11-Rad50-Nbs1 protein complex is one of the first responders to DNA double strand breaks. Studies have shown that the catalytic activities of the evolutionarily conserved Mre11-Rad50 (MR) core complex depend on an ATP-dependent global conformational change that takes the macromolecule from an open, extended structure in the absence of ATP to a closed, globular structure when ATP is bound. We have previously identified an additional ‘partially open’ conformation using Luminescence Resonance Energy Transfer (LRET) experiments. Here, a combination of LRET and the molecular docking program HADDOCK was used to further investigate this partially open state and identify three conformations of MR in solution: closed, partially open, and open, which are in addition to the extended, apo conformation. Mutants disrupting specific Mre11-Rad50 interactions within each conformation were used in nuclease activity assays on a variety of DNA substrates to help put the three states into a functional perspective. LRET data collected on MR bound to DNA demonstrate that the three conformations also exist when nuclease substrates are bound. These models were further supported with SAXS data which corroborate the presence of multiple states in solution. Together, the data suggest a mechanism for the nuclease activity of the MR complex along the DNA.