Can the smaller response to D b NPN3A be correlated with structural constraints or any decrease in stability for the pMHC-I complex? The D b NPN3A crystal structure containing the heavy chain of H-2D b (residues 1-275), the b2 microglobulin (residues 1-99) and the 9 residues of the NPN3A peptide was determined to a 2.6 Å resolution ( Fig. 3 and Table 1 ), with a final R factor of 22.1% and an R free of 30.4%. The structure of the H-2D b -NPN3A complex was compared (Fig. 3B , Table 1 ) to the wt D b NP 366 [23] . As observed for the wt D b NP 366 , the mutant NPN3A peptide bound H-2D b in an extended conformation, the P2-Ser, P5-Asn and P9-Met represent the anchor residues, P3-Ala semi-anchor residue, whereas the P4-Glu, P6-Met, P7-Glu and P8-Thr are solvent exposed and available for contact by the TCR (Fig. 3AB) . However, the mutation of P3-Asn to Ala leads to a loss of 35 contacts between the peptide and the MHC molecule. In comparison to the wt NP 366 that makes 636 contacts with the H-2D b molecule, the NPN3A peptide achieves only 601 contacts. Interestingly, the Asn3 Ala mutation abolishes contacts between the P3 residue of the peptide with His155 and the Tyr156 of the MHC and eliminates one hydrogen bond between the P3-Asn Nd2 and the P4-Glu O (Fig. 3CD ). The loss of inter-residue bonding between 3N and 4E within the NPN3A peptide may also be important for TCR recognition as it changes the characteristics of the wt D b NP 366 epitope (defined as type III constraint) [30] into an unconstrained D b NPN3A epitope. This change of constraint within the epitope could affect the dynamics of the peptide upon TCR ligation, and subsequently alter TCR reactivity toward the mutated epitope. In addition, the loss of contacts with the His155 of the MHC is of interest, as position 155, termed the ''gate-keeper residue'' [31] is involved in contacting the TCRs in all TCRpMHC-I complexes solved to date [32] . Thus, the lack of interactions between the NPN3A peptide and the MHC through His155 may also affect recognition of the complex by D b NP 366specific TCRs.