The immunogenicity of H-2D(b) (D-b) restricted epitopes can be significantly increased by substituting peptide position 3 to a proline (p3P). The p3P modification enhances MHC stability without altering the conformation of the modified epitope allowing for T-cell cross-reactivity with the native peptide. The present study reveals how specific interactions between p3P and the highly conserved MHC heavy chain residue Y159 increase the stability of D-b in complex with an optimized version of the melanoma-associated epitope gp100(25-33). Furthermore, the p3P modification directly increased the affinity of the D-b/gp100(25-33)-specific T-cell receptor (TCR) pMel. Surprisingly, the enhanced TCR binding was independent from the observed increased stability of the optimized D-b/gp100(25-33) complex and from the interactions formed between p3P and Y159, indicating a direct effect of the p3P modification on TCR recognition.