Distinct CDR3 conformations in TCRs determine the level of cross-reactivity for diverse antigens, but not the docking orientation

T cells are known to cross-react with diverse peptide MHC Ags through their αβ TCR. To explore the basis of such cross-reactivity, we examined the 2C TCR that recognizes two structurally distinct ligands, SIY-K^b and alloantigen QL9-L^d. In this study we characterized the cross-reactivity of several high-affinity 2C TCR variants that contained mutations only in the CDR3α loop. Two of the TCR lost their ability to cross-react with the reciprocal ligand (SIY-K^b), whereas another TCR (m67) maintained reactivity with both ligands. Crystal structures of four of the TCRs in complex with QL9-L^d showed that CDR1, CDR2, and CDR3β conformations and docking orientations were remarkably similar. Although the CDR3α loop of TCR m67 conferred a 2000-fold higher affinity for SIY-K ^b, the TCR maintained the same docking angle on QL9-L^d as the 2C TCR. Thus, CDR3α dictated the affinity and level of cross-reactivity, yet it did so without affecting the conserved docking orientation.