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.
