Understanding HLA-A*02 Polymorphisms by NMR: Structural and Dynamic Insights into Peptide Presentation and TCR Recognition

Major histocompatibility complex class I (MHC-I) molecules present intracellular peptides to T cell receptors (TCRs), enabling immune surveillance and cytotoxic T cell responses. Human leukocyte antigen (HLA) polymorphisms critically influence this process and despite extensive characterization of HLA-A*0201, the structural and functional consequences of micropolymorphisms within closely related HLA-A*02 alleles in modulating peptide presentation and TCR engagement remains poorly understood. Here, we investigate how subtle amino acid differences in HLA-A*0204, HLA-A*0206, and HLA-A*0207 influence peptide presentation and TCR engagement. Our aims were to compare these alleles to the well-characterized HLA- A*0201, elucidate how peptide variation drives immunogenicity, quantify allele-specific differences in TCR binding, and establish a recombinant strategy for peptide production. To achieve this, we combined in vitro refolding of peptide-MHC-I complexes, peptide-specific analyses, and TCR titration experiments with structural characterisation using Nuclear Magnetic Resonance (NMR) spectroscopy. Specifically, 15N-TROSY-HSQC experiments were used to monitor changes in the protein backbone, while 13C-SOFAST-HMQC experiments provided information on methyl-containing side chains, enabling residue-specific assessment of structural and dynamic changes. The structural mapping revealed that single β-sheet mutations in HLA-A*0204, -0206, and -0207, relative to -0201 remodel the peptide-binding groove and modulate exposure of the central residues, consistent with reported TCR affinity differences. Chemical shift perturbations (CSPs) localised primarily to the α1 and α2 helices, directly frame the peptide-binding cleft whilst a subset of CSPs was conserved across alleles, indicating shared structural adjustments despite polymorphic variation. Comparative peptide-bound analyses of HLA-A*0201 revealed that, while the global MHC-I fold remains intact, local conformational flexibility within the groove is peptide-dependent. The performed TCR titrations demonstrated canonical TCR-induced perturbations and dynamic allosteric effects on the peptide cleft. Collectively, our findings highlight how micropolymorphisms finely tune MHC-I structure, modify peptide presentation and TCR recognition. Beyond advancing mechanistic understanding, these insights provides a framework for rational peptide design and personalised immunotherapy strategies, emphasising allele-specific considerations in adaptive immune targeting.