Structure and function of the T-cell receptor: insights from X-ray crystallography.
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Crystallography, X-Ray↗
Biomedical subjects
R A Mariuzza
Publications and source records attributed to R A Mariuzza.
The structure of the T cell antigen receptor.
Recent crystallographic studies of T cell antigen receptor (TCR) fragments from the alpha and beta chains have now confirmed the expected structural similarity to corresponding immunoglobulin domains. Although the three-dimensional structure of a complete TCR alpha beta heterodimer has not yet been determined, these results support the view that the extracellular region should resemble an immunoglobulin Fab fragment with the antigen-binding site formed from peptide loops homologous to immunoglobulin complementarity-determining regions (CDR). These preliminary results suggest that CDR1 and CDR2 may be less variable in structure than their immunoglobulin counterparts, consistent with the idea that they may interact preferentially with the less polymorphic regions of the molecules of the major histocompatibility complex. The region on the variable beta domain responsible for superantigen recognition is analyzed in detail. The implications for T cell activation from the interactions observed between domains of the alpha and beta chains are also discussed in terms of possible dimerization and allosteric mechanisms.
Amino Acid Sequence↗
Crystal structure of the V alpha domain of a T cell antigen receptor.
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.
Animals↗
Superantigen binding to a T cell receptor beta chain of known three-dimensional structure.
The three-dimensional structure of an unglycosylated T cell antigen receptor (TCR) beta chain has recently been determined to 1.7 A resolution. To investigate whether this soluble beta chain (murine V beta 8.2J beta 2.1C beta 1) 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 beta 8.2. In contrast, SEA, which does not stimulate V beta 8.2-bearing cells, does not bind the recombinant beta chain. Binding of the beta 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 beta chain bound SEC1, 2, and 3 with KDs of 0.9-2.5 microM, the corresponding value for SEB was approximately 140 microM. 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 of the 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 beta chain and found that carbohydrate apparently does not contribute to recognition, even though the N-linked glycosylation sites at V beta 8.2 residues Asn24 and Asn74 are at or near the putative SAG-binding site. This result, along with the structural basis for the V beta specificity of SEs, are discussed in relation to the crystal structure of the unglycosylated beta chain.
Enterotoxins↗
Structure and thermodynamics of antigen recognition by antibodies.
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Amino Acid Sequence↗
Molecular basis of antigen mimicry by an anti-idiotope.
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.
Amino Acid Sequence↗
Crystal structure of the beta chain of a T cell antigen receptor.
The crystal structure of the extracellular portion of the beta chain of a murine T cell antigen receptor (TCR), determined at a resolution of 1.7 angstroms, shows structural homology to immunoglobulins. The structure of the first and second hypervariable loops suggested that, in general, they adopt more restricted sets of conformations in TCR beta chains than those found in immunoglobulins; the third hypervariable loop had certain structural characteristics in common with those of immunoglobulin heavy chain variable domains. The variable and constant domains were in close contact, presumably restricting the flexibility of the beta chain. This may facilitate signal transduction from the TCR to the associated CD3 molecules in the TCR-CD3 complex.
Amino Acid Sequence↗
Thermodynamics of antigen-antibody binding using specific anti-lysozyme antibodies.
Titration calorimetry measurements on the binding of hen lysozyme to the specific monoclonal IgG antibodies D1.3, D11.15, D44.1, F9.13.7, F10.6.6, their papain-cleaved antigen binding fragments (Fab) and their protein-engineered fragments consisting of non-covalently linked heavy variable chain and light variable chain domains (Fv) were performed between 6-50 degrees C in 0.15 M NaCl, 0.01 M sodium phosphate pH 7.1. The binding thermodynamic free energy change (delta G degrees b), enthalpy change (delta Hb), and entropy change (delta Sb) were the same for the whole IgG and its Fv and Fab fragments. With the exception of F9.13.7 at 13 degrees C, all the binding reactions were enthalpically driven with enthalpy changes ranging from -129 +/- 7 kJ mol-1 (D1.3 at 49.8 degrees C) to -26.2 +/- 0.6 kJ mol-1 (D44.1 at 8.0 degrees C). The heat capacity changes for the binding reaction (delta Cp) ranged from -2.72 +/- 0.16 kJ mol-1 K-1 (F9.13.7) to -0.95 +/- 0.06 kJ mol-1 K-1 (F10.6.6). The apolar surface areas buried at the binding sites estimated from the heat capacity changes indicate that the binding reactions are primarily hydrophobic, contrary to the mainly observed enthalpy-driven nature of the reactions. Conformational stabilization and the presence of water at the antigen-antibody interface may account for this discrepancy.
Animals↗
Protein motion and lock and key complementarity in antigen-antibody reactions.
Antibodies possess a highly complementary combining site structure to that of their specific antigens. In many instances their reactions are driven by enthalpic factors including, at least in the case of the reaction of monoclonal antibody D1.3 with lysozyme, enthalpy of solvation. They require minor structural rearrangements, and their equilibrium association constants are relatively high (10(7)-10(11) M-1). By contrast, in an idiotope--anti-idiotope (antibody-antibody) reaction, which is entropically driven, the binding equilibrium constant is only 1.5 x 10(5) M-1 at 20 degrees C. This low value results from a slow association rate (10(3) M-1 s-1) due to a selection of conformational states that allow one of the interacting molecular surfaces (the idiotope on antibody D1.3) to become complementary to that of the anti-idiotopic antibody. Thus, antibody D1.3 reacts with two different macromolecules: with its specific antigen, hen egg lysozyme, and with a specific anti-idiotopic antibody. Complementarity with lysozyme is closer to a "lock and key" model and results in high affinity (2-4 x 10(8) M-1). That with the anti-idiotopic antibody involves conformational changes at its combining site and it results in a lower association constant (1.5 x 10(5) M-1). Thus, an "induced fit" mechanism may lead to a broadening of the binding specificity but with a resulting decrease in the intrinsic binding affinity which may weaken the physiological function of antibodies.
Animals↗
Crystallization and preliminary X-ray diffraction study of an idiotope-anti-idiotope Fv-Fv complex.
A complex between the Fv fragment of an anti-hen eggwhite lysozyme antibody (D1.3) and the Fv fragment of an antibody specific for an idiotypic determinant of D1.3 has been crystallized in a form suitable for X-ray diffraction analysis. Both Fv fragments were expressed in soluble form in Escherichia coli and purified by affinity chromatography; diffraction-quality crystals were only obtained following separation of each Fv into distinct isoelectric forms. The crystals belong to space group C2, have unit cell dimensions a = 152.8 A, b = 79.4 A, c = 51.5 A, beta = 100.2 degrees, and diffract to better than 2.2 A resolution. The solvent content of the crystals is approximately 60% (v/v) with one Fv-Fv complex in the asymmetric unit. The ability to readily express both components of an antigen-antibody system in bacteria will allow us to rigorously assess the energetic contribution of individual amino acids to complex formation through pairwise mutagenesis of interacting residues.
Amino Acid Sequence↗
Crystallization and preliminary X-ray diffraction study of a bacterially produced T-cell antigen receptor V alpha domain.
A recombinant form of the variable domain of the alpha chain of a murine T-cell receptor specific for the N-terminal nonapeptide of myelin basin protein in association with the major histocompatibility complex class II I-Au molecule has been crystallized in a form suitable for X-ray diffraction analysis. This protein was secreted into the periplasmic space of Escherichia coli cells and affinity-purified using a nickel chelate adsorbent. The crystals are orthorhombic, space group P2(1)2(1)2, with unit cell dimensions a = 97.7 A, b = 79.6 A, c = 30.4 A and diffract to beyond 2.2 A resolution. The ability to crystallize a T-cell receptor domain produced in bacteria strongly suggests that the periplasmic space can provide a suitable environment for the correct in vivo folding of this class of antigen recognition molecules.
Animals↗
Solvent rearrangement in an antigen-antibody interface introduced by site-directed mutagenesis of the antibody combining site.
The three-dimensional structure of a site-directed mutant of the bacterially expressed Fv fragment from monoclonal antibody D1.3, complexed to the specific antigen lysozyme has been determined to a nominal resolution of 1.8 A using X-ray diffraction data. The replacement of VL Trp92 by Asp allows two water molecules to occupy space taken by Trp92 in the wild-type complex, in agreement with a previous observation that water molecules play an important role in stabilizing this antigen-antibody complex. The equilibrium constant for the binding of the mutant Fv to the antigen decreases by three orders of magnitude (from 2.3 x 10(8) M-1 to 2.6 x 10(5) M-1). Titration calorimetry shows that this results from a smaller negative binding enthalpy (delta delta H = -16 kJ mol-1 at 24 degrees C), whereas the value of the binding entropy is not affected. Since in the complex between the mutated Fv and antigen the buried area has decreased relative to that of the wild-type Fv by about 150 A2, the contribution of the buried unit area to the decrease in free energy (delta Gzero) is approximately 117 J mol-1 (28 cal mol-1) per A2. The loss of interatomic contacts in replacing Trp by Asp permits an approximate calculation for the contribution of van der Waals interactions made by Trp92 in this complex, which gives an average of 2.1 kJ mol-1 (0.5 kcal mol-1) for contacts between carbon atoms.
Antibodies, Monoclonal↗
Bound water molecules and conformational stabilization help mediate an antigen-antibody association.
We report the three-dimensional structures, at 1.8-A resolution, of the Fv fragment of the anti-hen egg white lysozyme antibody D1.3 in its free and antigen-bound forms. These structures reveal a role for solvent molecules in stabilizing the complex and provide a molecular basis for understanding the thermodynamic forces which drive the association reaction. Four water molecules are buried and others form a hydrogen-bonded network around the interface, bridging antigen and antibody. Comparison of the structures of free and bound Fv fragment of D1.3 reveals that several of the ordered water molecules in the free antibody combining site are retained and that additional water molecules link antigen and antibody upon complex formation. This solvation of the complex should weaken the hydrophobic effect, and the resulting large number of solvent-mediated hydrogen bonds, in conjunction with direct protein-protein interactions, should generate a significant enthalpic component. Furthermore, a stabilization of the relative mobilities of the antibody heavy- and light-chain variable domains and of that of the third complementarity-determining loop of the heavy chain seen in the complex should generate a negative entropic contribution opposing the enthalpic and the hydrophobic (solvent entropy) effects. This structural analysis is consistent with measurements of enthalpy and entropy changes by titration calorimetry, which show that enthalpy drives the antigen-antibody reaction. Thus, the main forces stabilizing the complex arise from antigen-antibody hydrogen bonding, van der Waals interactions, enthalpy of hydration, and conformational stabilization rather than solvent entropy (hydrophobic) effects.
Animals↗
Crystallization and preliminary X-ray diffraction analysis of the beta-chain of a T-cell antigen receptor.
A secreted form of the beta-chain of a T-cell receptor specific for a hemagglutinin peptide of influenza virus in the context of the major histocompatibility complex class II I-Ed molecule has been crystallized in a form suitable for X-ray diffraction analysis. The crystals are tetragonal, space group P4(1)2(1)2 (or P4(3)2(1)2), with cell dimensions a = b = 71.4 A, c = 312.9 A, and diffract to beyond 3.5 A resolution. The beta-chain appears to behave as a stable homodimer in solution.
Amino Acid Sequence↗
Structural and physicochemical analysis of the reaction between the anti-lysozyme antibody D1.3 and the anti-idiotopic antibodies E225 and E5.2.
The reaction between the mouse (BALB/c) anti-idiotopic monoclonal antibodies E225 and E5.2 and idiotopes on the (BALB/c) anti-lysozyme monoclonal antibody D1.3 has been characterized by titration calorimetry, by equilibrium sedimentation and by the determination of binding association and dissociation rates. The reaction between E5.2 and D1.3 is driven by a large negative enthalpy and its rate and equilibrium association constants are comparable to those observed in other antigen-antibody reactions. In contrast, the reaction between E225 and D1.3 is entropically driven and characterized by slow association kinetics (1 x 10(3) M-1 sec-1) and a resulting low equilibrium constant (Ka = 2 x 10(5) M-1). A correlation of these properties with the three-dimensional structure of the Fab225-FabD1.3 complex, previously determined by X-ray diffraction methods to 2.5 A resolution, indicates that conformational changes of several D1.3 contacting residues, located in its complementarity determining regions, may explain these features of the reaction.
Animals↗
The energetics of antigen-antibody binding.
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Animals↗
A soluble form of the human CD8 alpha chain expressed in the baculovirus system: biochemical characterization and binding to MHC class I.
We have generated a soluble form of the CD8 molecule consisting of the entire extracellular domains of the human alpha chain, by expressing a mutated CD8 alpha cDNA in SF9 cells infected with a recombinant baculovirus. The truncated molecule was secreted into the medium mostly as a disulfide-linked homodimer in which a single cysteine residue in the hinge-like region (Cys143) was sufficient to assure covalent bonding. Soluble CD8 purified to homogeneity appears to be monodisperse as assessed by gel filtration analysis and contains only O-linked carbohydrates. To determine whether recombinant CD8 can interact with MHC class I molecules, we developed an assay that measures binding of MHC class I-bearing cell lines to purified CD8 adsorbed to plastic plates. The level of binding of cells to immobilized CD8 depended on the amount of CD8 bound to the plate and correlated with the levels of cell surface MHC class I expression. The binding was specifically inhibited by monoclonal antibodies directed either against CD8 or MHC class I molecules. This assay therefore provides a way to measure CD8 binding to MHC class I independently of other cell-cell interactions and should allow direct structure-function studies.
Baculoviridae↗
The basics of binding: mechanisms of antigen recognition and mimicry by antibodies.
New insights into the nature of antigen-antibody recognition have been gained through X-ray crystallographic studies of immune complexes. In particular, it has been demonstrated that water molecules form an extended network bridging antigen and antibody, and are essential in achieving shape and chemical complementarity between their interacting surfaces. This finding has important implications for the energetics of the association reaction. Recently, X-ray data on the complex between a peptide hormone and an anti-anti-idiotypic antibody have been obtained. This has relevance to the structural basis of antigen mimicry by antibodies. The conformation of the bound peptide was found to be very similar to that of an antibody complementarity determining region loop, providing a direct structural explanation for how antigen mimicry by anti-idiotypic antibodies might occur.
Animals↗