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Dissociation of β2m from MHC Class I Triggers Formation of Noncovalent, Transient Heavy Chain Dimers

View ORCID ProfileCindy Dirscherl, Sara Löchte, View ORCID ProfileZeynep Hein, View ORCID ProfileJanine-Denise Kopicki, Antonia Regina Harders, Noemi Linden, Julian Weghuber, Maria Garcia-Alai, View ORCID ProfileCharlotte Uetrecht, Martin Zacharias, View ORCID ProfileJacob Piehler, View ORCID ProfilePeter Lanzerstorfer, View ORCID ProfileSebastian Springer
doi: https://doi.org/10.1101/2021.07.02.450866
Cindy Dirscherl
1Department of Life Sciences and Chemistry, Jacobs University Bremen, Germany
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Sara Löchte
2Department of Biology and Center of Cellular Nanoanalytics, University of Osnabrück, 49076 Osnabrück, Germany
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Zeynep Hein
1Department of Life Sciences and Chemistry, Jacobs University Bremen, Germany
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Janine-Denise Kopicki
3Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany
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Antonia Regina Harders
1Department of Life Sciences and Chemistry, Jacobs University Bremen, Germany
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Noemi Linden
1Department of Life Sciences and Chemistry, Jacobs University Bremen, Germany
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Julian Weghuber
4University of Applied Sciences Upper Austria, 4600 Wels, Austria
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Maria Garcia-Alai
5European Molecular Biology Laboratory, Hamburg Outstation, Hamburg, Germany
6Centre for Structural Systems Biology, Hamburg, Germany
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Charlotte Uetrecht
3Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany
7European XFEL, Schenefeld, Germany
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Martin Zacharias
8Physics Department, Technical University of Munich, Garching, Germany
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Jacob Piehler
2Department of Biology and Center of Cellular Nanoanalytics, University of Osnabrück, 49076 Osnabrück, Germany
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Peter Lanzerstorfer
6Centre for Structural Systems Biology, Hamburg, Germany
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Sebastian Springer
1Department of Life Sciences and Chemistry, Jacobs University Bremen, Germany
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  • For correspondence: s.springer@jacobs-university.de
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Abstract

Previously, we demonstrated that major histocompatibility complex class I molecules (MHC-I) that lose the antigenic peptide and the light chain beta-2 microglobulin (β2m) to become free heavy chains (FHCs) associate to form dimers or higher-order oligomers in the plasma membrane (Dirscherl et al., 2018). Here, we investigate this homotypic interaction of MHC-I FHCs by combining a micropattern assay with fluorescence recovery after photobleaching (FRAP) and with single molecule co-tracking to elucidate their molecular structure, abundance, and dynamics. We find that MHC-I FHC complexes are dimeric, transient, non-covalent, and mediated by the α3 domain. FHC interaction correlates with a decrease in the diffusion coefficient and an increase in the number of immobile molecules at the cell surface. Molecular docking and dynamics simulations suggest that in the complexes, the α3 domain of one FHC binds to another FHC in a manner similar to the β2m light chain. We propose distinct functions of the MHC-I FHC dimers.

Significance Statement MHC class I molecules are cell surface transmembrane proteins. In addition to binding receptors on other cells (in trans), such as the T cell receptor or inhibitory receptors of Natural Killer cells, they also bind to proteins on the same cell (in cis), including other class I molecules. The functions of such in cis associations are not well understood, and investigation is difficult because class I molecules exist on the cell membrane in three different states, depending on whether they are associated with their light chain and with peptide. Using three independent approaches, we report here the binding of class I free heavy chains (but not the other states) to each other in cis to form non-covalent dimers at the cell surface. The roles of these dimers can now be investigated; we propose that they function in signaling, endocytic sorting, or both.

Competing Interest Statement

The authors have declared no competing interest.

  • Abbreviations

    AUC
    area under the curve
    β2m
    beta-2 microglobulin
    BFA
    brefeldin A
    BSA
    buried surface area
    Db
    the murine MHC-I molecule H-2Db
    ER
    endoplasmic reticulum
    FHC
    free heavy chain
    FRAP
    fluorescence recovery after photobleaching
    GFP
    green fluorescent protein
    HA
    hemagglutinin (tag)
    HC
    heavy chain
    Kb
    the murine MHC-I molecule H-2Kb
    MD
    molecular dynamics
    MHC-I
    major histocompatibility complex class I
    MS
    mass spectrometry
    RMSD
    root mean square deviation
    SL8
    ligand peptide cognate to Kb (single-letter amino acid code SIINFEKL)
    SMCT
    single-molecule co-tracking
    SMT
    single-molecule tracking
    TAP
    transporter associated with antigen processing
    TIRF
    total internal reflection fluorescence
    TMD
    transmembrane domain.
  • Copyright 
    The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
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    Posted July 03, 2021.
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    Dissociation of β2m from MHC Class I Triggers Formation of Noncovalent, Transient Heavy Chain Dimers
    Cindy Dirscherl, Sara Löchte, Zeynep Hein, Janine-Denise Kopicki, Antonia Regina Harders, Noemi Linden, Julian Weghuber, Maria Garcia-Alai, Charlotte Uetrecht, Martin Zacharias, Jacob Piehler, Peter Lanzerstorfer, Sebastian Springer
    bioRxiv 2021.07.02.450866; doi: https://doi.org/10.1101/2021.07.02.450866
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    Dissociation of β2m from MHC Class I Triggers Formation of Noncovalent, Transient Heavy Chain Dimers
    Cindy Dirscherl, Sara Löchte, Zeynep Hein, Janine-Denise Kopicki, Antonia Regina Harders, Noemi Linden, Julian Weghuber, Maria Garcia-Alai, Charlotte Uetrecht, Martin Zacharias, Jacob Piehler, Peter Lanzerstorfer, Sebastian Springer
    bioRxiv 2021.07.02.450866; doi: https://doi.org/10.1101/2021.07.02.450866

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