Superantigens (SAgs) are microbial toxins defined by their ability to activate T lymphocytes in a T cell receptor (TCR)␤-chain variable domain (V␤)-specific manner. Although existing structural information indicates that diverse bacterial SAgs all uniformly engage the V␤ second complementarity determining region (CDR2␤) loop, the molecular rules that dictate SAg-mediated T cell activation and V␤ specificity are not fully understood. Herein we report the crystal structure of human V␤2.1 (hV␤2.1) in complex with the toxic shock syndrome toxin-1 (TSST-1) SAg, and mutagenesis of hV␤2.1 indicates that the non-canonical length of CDR2␤ is a critical determinant for recognition by TSST-1 as well as the distantly related SAg streptococcal pyrogenic exotoxin C. Frame work (FR) region 3 is uniquely critical for TSST-1 function explaining the fine V␤-specificity exhibited by this SAg. Furthermore, domain swapping experiments with SAgs, which use distinct domains to engage both CDR2␤ and FR3/4␤ revealed that the CDR2␤ contacts dictate T lymphocyte V␤-specificity. These findings demonstrate that the TCR CDR2␤ loop is the critical determinant for functional recognition and V␤-specificity by diverse bacterial SAgs.Antigen-specific T cell-mediated immunity is initiated through the interaction of the ␣␤ T cell receptor (TCR) 3 with a processed peptide antigen presented within the distal groove of self major histocompatibility (pMHC) complexes. A general consensus for typical TCR-pMHC recognition is that germ line encoded complementarity determining region loops (CDR) 1 and CDR2 of both the TCR ␣-and ␤-chains primarily recognize the ␣-helices of self pMHC complexes, while peptide specificity is mainly driven by the CDR3 loops (1, 2). Bacterial superantigens (SAgs) are potent immunostimulatory toxins that distort the molecular 'rules' which dictate normal TCR-pMHC class II (pMHC II) interactions. SAgs function by binding to lateral surfaces on both TCR ␤-chain variable domains (V␤) (3) and pMHC II complexes (4), resulting in the activation of T cells in a V␤-restricted manner (5). In the standard TCR-SAg-pMHC II model, the TCR ␤-chain, and pMHC II ␣-chain are wedged apart by the SAg such that the CDR3 loops of the ␣␤ TCR cannot interact directly with the antigenic peptide contained within the pMHC II complexes (6). Because SAgs bind the germ line encoded V␤ region, while essentially ignoring the highly diverse CDR3␤ loop, the frequency of T cells responding to SAg exposure exceeds that of conventional peptide antigens by many orders of magnitude. Through this mechanism, SAgs may ultimately mediate a cytokine storm disease known as the toxic shock syndrome (7).The bacterial pathogens Staphylococcus aureus and Streptococcus pyogenes are each capable of producing multiple distinct bacterial SAgs that fall into at least five distinct evolutionary groups, designated as I through V (Fig. 1A) (7, 8), that share a conserved three-dimensional fold (9) (Fig. 1B). Despite this conserved structure, SAgs can bind to pMHC II through diverse m...

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