| Summary: | Through major histocompatibility complex class I leader sequence-derived peptide (VL9) binding and CD94/NKG2 receptor engagement, human leucocyte antigen E (HLA-E) reports cellular health to NK cells. Previous studies demonstrated a strong bias for VL9 binding by HLA-E, a preference subsequently supported by structural analyses. However, Mycobacteria tuberculosis (Mtb) infection and Rhesus cytomegalovirus-vectored SIV vaccination revealed contexts where HLA-E and its rhesus orthologue, Mamu-E, present sequence-diverse pathogen-derived peptides to CD8+ T cells, respectively. In light of unprecedented vaccine-induced Mamu-E-restricted CD8+ T cell-mediated protection against SIV, it is now important to re-explore the peptide binding specificity of the human orthologue, HLA-E, from the view of developing a universal HLA-E-targeted HIV vaccination strategy in humans. Through the development and optimisation of a high throughput in vitro peptide binding assay, we demonstrated an HLA-E binding capacity for a number of pathogen-derived epitopes containing non-canonical anchor residues. As previous crystal structures of HLA-E were exclusively solved in complex with canonical VL9 peptides, we also investigated the structural basis underlying pathogen-derived peptide binding. We obtained a panel of HLA-E crystal structures in complex with a number of Mtb-derived epitopes in addition to the HIV counterpart peptide to the vaccine-identified SIV Gag ‘supertope69’. Strikingly, despite the presence of canonical primary anchor residues, the HIV peptide adopted an alternative conformational motif within the HLA-E peptide binding groove, deviating from that of canonical VL9 and in turn generating a distinct solvent exposure profile, with potential implications for T cell receptor recognition and immunogenicity. Further, combined structural and mutagenesis analyses illustrated a greater tolerance for hydrophobic and polar residues in the primary pockets of the HLA-E binding groove than previously anticipated. Finally, biophysical analyses demonstrated the impact of suboptimal peptide binding on the overall conformational ensemble of HLA-E complexes in solution.
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