Superantigens (SAgs) are viral or bacterial proteins that act as potent T-cell stimulants and have been implicated in a number of human diseases, including toxic shock syndrome, diabetes mellitus and multiple sclerosis. The interaction of SAgs with the T-cell receptor (TCR) and major histocompatibility complex (MHC) proteins results in the stimulation of a disproportionately large fraction of the T-cell population. We report here the crystal structures of the beta-chain of a TCR complexed with the Staphylococcus aureus enterotoxins C2 and C3 (SEC2, SEC3). These enterotoxins, which cause both toxic shock and food poisoning, bind in an identical way to the TCR beta-chain. The complementarity-determining region 2 (CDR2) of the beta-chain and, to lesser extents, CDR1 and hypervariable region 4 (HV4), bind in a cleft between the two domains of the SAgs. Thus, there is considerable overlap between the SAg-binding site and the peptide/MHC-binding sites of the TCR. A model of a TCR-SAg-MHC complex constructed from the crystal structures of (1) the beta-chain-SEC3 complex, (2) a complex between staphylococcal enterotoxin B (SEB) and an MHC molecule, and (3) a TCR V(alpha) domain, reveals that the SAg acts as a wedge between the TCR and MHC to displace the antigenic peptide away from the TCR combining site. In this way, the SAg is able to circumvent the normal mechanism for T-cell activation by specific peptide/MHC complexes.

The crystal structure of the V alpha domain of a T cell antigen receptor (TCR) was determined at a resolution of 2.2 angstroms. This structure represents an immunoglobulin topology set different from those previously described. A switch in a polypeptide strand from one beta sheet to the other enables a pair of V alpha homodimers to pack together to form a tetramer, such that the homodimers are parallel to each other and all hypervariable loops face in one direction. On the basis of the observed mode of V alpha association, a model of an (alpha beta)2 TCR tetramer can be positioned relative to the major histocompatibility complex class II (alpha beta)2 tetramer with the third hypervariable loop of V alpha over the amino-terminal portion of the antigenic peptide and the corresponding loop of V beta over its carboxyl-terminal residues. TCR dimerization that is mediated by the alpha chain may contribute to the coupling of antigen recognition to signal transduction during T cell activation.

Idiotopes are antigenic determinants, unique to an antibody or group of antibodies, defined by the reaction of anti-idiotopic antibodies with the antibodies bearing the idiotopes. The ensemble of idiotopes of an antibody constitutes its idiotype. Idiotypes are useful as markers to follow specific antibodies and clones of cells in immune responses and the inheritance of immunoglobulin genes. As external antigens and anti-idiotypic antibodies can competitively bind the combining site of specific antibodies, some anti-idiotypic antibodies may resemble the external antigen, thus mimicking its structure. It has been proposed that an anti-idiotypic antibody, anti-anti-X, may resemble the external antigen X and thus carry its 'internal image', but this idea is not unequivocally supported by the three-dimensional structures of anti-idiotopic antibodies, either because the structures of the external antigen or of the anti-idiotopic antibody were unknown, or because the anti-idiotopic antibodies showed no resemblance to the external antigens (reviewed in ref. 10). Functional mimicry of ligands of biological receptors by anti-idiotypic antibodies has been described in several systems (reviewed in ref. 11). But how closely can antibodies mimic antigens at the molecular level? Here we present the crystal structure of an idiotope-anti-idiotope complex between the Fv fragments of the anti-lysozyme antibody D1.3 and the anti-D1.3 antibody E5.2. D1.3 contacts the antigen, lysozyme and the anti-idiotopic E5.2 through essentially the same combining-site residues. In addition, E5.2 interacts with D1.3, making contacts similar to those between lysozyme and D1.3. Thus, the anti-idiotopic antibody E5.2 mimics lysozyme in its binding interactions with D1.3. Validating these observations, E5.2, used as an immunogen, induces an anti-lysozyme response.

Antigen-antibody complexes provide useful models for analyzing the thermodynamics of protein-protein association reactions. We have employed site-directed mutagenesis, X-ray crystallography, and isothermal titration calorimetry to investigate the role of hydrophobic interactions in stabilizing the complex between the Fv fragment of the anti-hen egg white lysozyme (HEL) antibody D1.3 and HEL. Crystal structures of six FvD1.3-HEL mutant complexes in which an interface tryptophan residue (V(L)W92) has been replaced by residues with smaller side chains (alanine, serine, valine, aspartate, histidine, and phenylalanine) were determined to resolutions between 1.75 and 2.00 A. In the wild-type complex, V(L)W92 occupies a large hydrophobic pocket on the surface of HEL and constitutes an energetic "hot spot" for antigen binding. The losses in apolar buried surface area in the mutant complexes, relative to wild-type, range from 25 (V(L)F92) to 115 A(2) (V(L)A92), with no significant shifts in the positions of protein atoms at the mutation site for any of the complexes except V(L)A92, where there is a peptide flip. The affinities of the mutant Fv fragments for HEL are 10-100-fold lower than that of the original antibody. Formation of all six mutant complexes is marked by a decrease in binding enthalpy that exceeds the decrease in binding free energy, such that the loss in enthalpy is partly offset by a compensating gain in entropy. No correlation was observed between decreases in apolar, polar, or aggregate (sum of the apolar and polar) buried surface area in the V(L)92 mutant series and changes in the enthalpy of formation. Conversely, there exist linear correlations between losses of apolar buried surface and decreases in binding free energy (R(2) = 0.937) as well as increases in the solvent portion of the entropy of binding (R(2) = 0.909). The correlation between binding free energy and apolar buried surface area corresponds to 21 cal mol(-1) A(-2) (1 cal = 4.185 J) for the effective hydrophobicity at the V(L)92 mutation site. Furthermore, the slope of the line defined by the correlation between changes in binding free energy and solvent entropy approaches unity, demonstrating that the exclusion of solvent from the binding interface is the predominant energetic factor in the formation of this protein complex. Our estimate of the hydrophobic contribution to binding at site V(L)92 in the D1.3-HEL interface is consistent with values for the hydrophobic effect derived from classical hydrocarbon solubility models. We also show how residue V(L)W92 can contribute significantly less to stabilization when buried in a more polar pocket, illustrating the dependence of the hydrophobic effect on local environment at different sites in a protein-protein interface.

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SummaryThe three-dimensional structure of an unglycosylated T cell antigen receptor (TCR) R chain has recently been determined to 1 .7 A resolution. To investigate whether this soluble R chain (murine V(38.2JR2 .IC(31) retains superantigen (SAG)-binding activity, we measured its affinity for various bacterial SAGS in the absence of MHC class II molecules . Dissociation constants (KDs) were determined using two independent techniques : surface plasmon resonance detection and sedimentation equilibrium. Specific binding was demonstrated to staphylococcal enterotoxins (SEs) B, C1, C2, and C3 and to streptococcal pyrogenic exotoxin A (SPEA), consistent with the known proliferative effects of these SAGs on T cells expressing V(38.2. In contrast, SEA, which does not stimulate V(38.2-bearing cells, does not bind the recombinant R chain. Binding of the R chain to SAGs was characterized by extremely fast dissociation rates (>0 .1 s-1), similar to those reported for certain leukocyte adhesion molecules . Whereas the R chain bound SEC1, 2, and 3 with KDs of 0.9-2.5 [LM, the corresponding value for SEB was^140 p,M. The much weaker binding to SEB than to SEC1, 2, or 3 was surprising, especially since SEB was found to actually be 3-to 10-fold more effective, on a molar basis, than the other toxins in stimulating the parental T cell hybridoma . We interpret these results in terms ofthe ability of SEC to activate T cells independently of MHC, in contrast to SEB . We have also measured SE binding to the glycosylated form of the R chain and found that carbohydrate apparently does not contribute to recognition, even though the N-linked glycosylation sites at V(38.2 residues Asn24 and Asn74 are at or near the putative SAG-binding site. This result, along with the structural basis for the V(3 specificity of SEs, are discussed in relation to the crystal structure of the unglycosylated R chain .Antigen recognition by T lymphocytes is mediated by highly diverse cell surface glycoproteins known as T cell receptors (TCRs)t . In addition to recognizing peptide antigens complexed with products of the MHC complex, TCRs interact with a class of molecules known as superantigens (SAGs), which stimulate T cells bearing particular V(3 'Abbreviations used in this paper :

Antigen recognition by T lymphocytes is mediated by cell-surface glycoproteins known as T-cell antigen receptors (TCRs). These are composed of alpha and beta, or gamma and delta, polypeptide chains with variable (V) and constant (C) regions. In contrast to alphabeta TCRs, which recognize antigen only as peptide fragments bound to molecules of the major histocompatibility complex (MHC), gammadelta TCRs appear to recognize proteins directly, without antigen processing, and to recognize MHC molecules independently of the bound peptide. Moreover, small phosphate-containing non-peptide compounds have also been identified as ligands for certain gammadelta T cells. These studies indicate that antigen recognition by gammadelta TCRs may be fundamentally different from that by alphabeta TCRs. The three-dimensional structures of several alphabeta TCRs and TCR fragments, and their complexes with peptide-MHC or superantigens, have been determined. Here we report the crystal structure of the Vdelta domain of a human gammadelta TCR at 1.9 A resolution. A comparison with antibody and alphabeta TCR V domains reveals that the framework structure of Vdelta more closely resembles that of VH than of Valpha, Vbeta or VL (where H and L refer to heavy and light chains), whereas the relative positions and conformations of its complementarity-determining regions (CDRs) share features of both Valpha and VH. These results provide the first direct evidence that gammadelta TCRs are structurally distinct from alphabeta TCRs and, together with the observation that the CDR3 length distribution of TCR delta chains is similar to that of immunoglobulin heavy chains, are consistent with functional studies suggesting that recognition of certain antigens by gammadelta TCRs may resemble antigen recognition by antibodies.

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