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

At the plasma membrane of mammalian cells, major histocompatibility complex class I molecules (MHC-I) present antigenic peptides to cytotoxic T cells. Following the loss of the peptide and the light chain beta-2 microglobulin ({beta}2m), the resulting free heavy chains (FHCs) can associate into homotypic complexes in the plasma membrane. Here, we investigate the stoichiometry and dynamics of MHC-I FHCs assemblies by combining a micropattern assay with fluorescence recovery after photobleaching (FRAP) and with single molecule co-tracking. We identify non-covalent MHC-I FHC dimers mediated by the 3 domain as the prevalent species at the plasma membrane, leading a moderate decrease in the diffusion coefficient. MHC-I FHC dimers show increased tendency to cluster into higher order oligomers as concluded from an increased immobile fraction with higher single molecule co-localization. In vitro studies with isolated proteins in conjunction with molecular docking and dynamics simulations suggest that in the complexes, the 3 domain of one FHC binds to another FHC in a manner similar to the {beta}2m light chain. Significance StatementMHC class I molecules are cell surface transmembrane proteins with key functions in adaptive immunity against viral infections. The spatiotemporal organization of fully assembled MHC I at the cell surface and its function with respect to trans-interactions with T and NK cells has been studied in detail. By contrast, the consequences of peptide and {beta}2m dissociation yielding to formation of free heavy chains (FHC) have remained unclear. We have discovered that class I free heavy chains form distinct non-covalent dimers at the cell surface rather than non-specific clustering, and we have identified a dimerization interface mediated by the 3 domain. We propose that these non-covalent dimers are the basis of distinct signaling and endocytic sorting of MHC I FHC. This is to be explored in further work.