Visualization and characterization of the elusive MHC I intermediate states by x-ray crystallography and in silico studies

Abstract Background: Studies have suggested that MHC class I (MHC I) molecules fluctuate rapidly between conformational states as they sample peptides for potential ligands. It is the molecular features of these intermediates that are recognized by tapasin and TAPBPR at the C-terminus of the groove. To date, however, MHC I intermediates remain elusive molecules due to their transient nature. Moreover, whether the N-terminus of the groove has a role in MHC I maturation, and if it also serves as a conformationally sensing region, is not well characterized. Methods: We designed the HLA-B8-restricted 20mer (RA) 6FAKKKYCL and (RA) 6FVKKKYCL peptides and corresponding 8mer FAKKKYCL and FVKKKYCL controls. We determined the x-ray crystal structures of HLA-B8E76C presenting these four peptides. Molecular dynamics (MD) simulations of the complexes were also performed. Results: The structures of FA and FV 20mers revealed significant conformational heterogeneity at the N-terminus of the groove. Long stretches of N-terminal MHC I residues were missing in the electron density maps creating an unstructured and widely open-ended groove. Our structures also showed highly unusual features in MHC I and peptides at the N-terminus of the groove, and in MHC I-peptide interaction. MD simulations showed that these complexes have varying degrees of mobility in a manner consistent with our structures. We suggest that our 20mer structures represent conformational substates explored by MHC I during peptide selection. Conclusion: Our study advances our understanding of peptide-induced conformational adaptation and functional dynamics in MHC I, and brings into focus the N-terminus of the groove as an important region in mechanisms of high-affinity peptide selection. NIAID, National Institutes of Health (R01 AI114467, R01 AI108546, and R21 AI173863)