MHC class I protein dynamics and antigen presenting function

Abstract

MHC class I molecules on the cell surface present endogenous peptide to the adaptive immune system. The mature complex is trimeric, consisting of an α-chain encoding membrane-distal peptide binding and membrane-bound α3 domains, an invariant β2m protein, and a peptide. The α-chain is highly polymorphic, with the six alleles expressed by an individual capable of presenting all potential antigens due to promiscuous peptide binding. In addition to presenting antigen, MHC class I has a role in selecting high-affinity peptides during complex maturation within the endoplasmic reticulum. There is a large pool of available peptides, but only highaffinity interactions are capable of eliciting an immune response. Whilst the structure of the mature complex has been well documented by X-ray crystallography, the process of peptide optimisation remains unclear. It is apparent that a conformational change within the peptide binding domain is required to exchange low-affinity for high-affinity antigen. Molecular dynamics simulations have been employed to model the dynamics of MHC class I but have not yet been verified by direct observation of such motion. This thesis investigates the dynamics of MHC class I using nuclear magnetic resonance (NMR) spectroscopy, focusing on the possible existence of alternative conformations within the peptide binding domain that permit peptide exchange. Protein production methods have been established for reliable generation of mature MHC class I, consisting of the murine H-2Db α-chain allele, human β2m, and the high affinity FAPGNYPAL peptide. Following assignment of backbone resonances, NMR analyses include residual dipolar coupling data revealing the potential for up to a 20° twist between the peptide binding and membrane-proximal domains. Heteronuclear relaxation data shows that MHC class I is inherently dynamic, experiencing multiple conformations in regions throughout the secondary structure. Mobility is largely located within the peptide binding domain and provides confirmation of predictions from molecular dynamics. This thesis confirms that MHC class I is capable of forming an intermediate conformation during peptide binding, during which it is peptide-receptive.

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