Molecular Basis of TCR Selectivity, Cross-Reactivity, and Allelic Discrimination by a Bacterial Superantigen: Integrative Functional and Energetic Mapping of the SpeC-Vβ2.1 Molecular Interface

Abstract

Superantigens activate large fractions of T cells through unconventional interactions with both TCR β-chain V domains (Vβs) and MHC class II molecules. The bacterial superantigen streptococcal pyrogenic exotoxin C (SpeC) primarily stimulates human Vβ2+ T cells. Herein, we have analyzed the SpeC-Vβ2.1 interaction by mutating all SpeC residues that make contact with Vβ2.1 and have determined the energetic and functional consequences of these mutations. Our comprehensive approach, including mutagenesis, functional readouts from both bulk T cell populations, and an engineered Vβ2.1+ Jurkat T cell, as well as surface plasmon resonance binding analysis, has defined the SpeC “functional epitope” for TCR engagement. Although only two SpeC residues (Tyr15 and Arg181) are critical for activation of virtually all human CD3+ T cells, a larger cluster of four hot spot residues are required for interaction with Vβ2.1. Three of these residues (Tyr15, Phe75, and Arg181) concentrate their binding energy on the CDR2 loop residue Ser52a, a noncanonical residue insertion found only in Vβ2 and Vβ4 chains. Plasticity of this loop is important for recognition by SpeC. Although SpeC interacts with the Vβ2.1 hypervariable CDR3 loop, our data indicate these contacts have little to no influence on the functional interaction with Vβ2.1. These studies also provide a molecular basis for selectivity and cross-reactivity of SpeC-TCR recognition and reveal a degree of fine specificity in these interactions, whereby certain SpeC mutants are capable of distinguishing between different alleles of the same Vβ domain subfamily.