Dissociation of β2m from cell surface MHC class I triggers formation of noncovalent, transient heavy chain dimers

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 at the cell surface are known to associate, forming oligomers on the plasma membrane that are insufficiently understood. Here, we investigate the homotypic interaction of MHC I free heavy chains by combining a printed antibody micropattern assay with fluorescence recovery after photobleaching (FRAP) and with single molecule co-tracking in order to elucidate their molecular structure, abundance, and dynamics. We find that MHC I free heavy chain complexes are dimeric, transient, non-covalent, and mediated by the α3 domain. Free heavy chain 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 free heavy chain in a manner similar to the β2m light chain. We propose distinct functions of the MHC I free heavy chain dimers in signaling and in endocytic sorting.