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R A Mariuzza

Publications and source records attributed to R A Mariuzza.

At least 19 recordsLinked to original sources
PubMed1999

Role of the T cell receptor alpha chain in stabilizing TCR-superantigen-MHC class II complexes.

Superantigens (SAGs) activate T cells by simultaneously binding the Vbeta domain of the TCR and MHC class II molecules on antigen-presenting cells. The preferential expression of certain Valpha regions among SAG-reactive T cells has suggested that the TCR alpha chain may modulate the level of activation through an interaction with MHC. We demonstrate that the TCR alpha chain is required for maximum stabilization of the TCR-SAG-MHC complex and that the alpha chain increases the half-life of the complex to match those of TCR-peptide/MHC complexes. The site on the TCR alpha chain responsible for these effects is CDR2. Thus, the overall stability of the TCR-SAG-MHC complex is determined by the combination of three distinct interactions: TCR-SAG, SAG-MHC, and MHC-TCR.

Amino Acid Substitution↗
PubMed1999

The structural basis of T cell activation by superantigens.

Superantigens (SAGs) are a class of immunostimulatory and disease-causing proteins of bacterial or viral origin with the ability to activate large fractions (5-20%) of the T cell population. Activation requires simultaneous interaction of the SAG with the V beta domain of the T cell receptor (TCR) and with major histocompatibility complex (MHC) class II molecules on the surface of an antigen-presenting cell. Recent advances in knowledge of the three-dimensional structure of bacterial SAGs, and of their complexes with MHC class II molecules and the TCR beta chain, provide a framework for understanding the molecular basis of T cell activation by these potent mitogens. These structures along with those of TCR-peptide/MHC complexes reveal how SAGs circumvent the normal mechanism for T cell activation by peptide/MHC and how they stimulate T cells expressing TCR beta chains from a number of different families, resulting in polyclonal T cell activation. The crystal structures also provide insights into the basis for the specificity of different SAGs for particular TCR beta chains, and for the observed influence of the TCR alpha chain on SAG reactivity. These studies open the way to the design of SAG variants with altered binding properties for TCR and MHC for use as tools in dissecting structure-activity relationships in this system.

Amino Acid Sequence↗
PubMed1998

A mutational analysis of binding interactions in an antigen-antibody protein-protein complex.

Alanine scanning mutagenesis, double mutant cycles, and X-ray crystallography were used to characterize the interface between the anti-hen egg white lysozyme (HEL) antibody D1.3 and HEL. Twelve out of the 13 nonglycine contact residues on HEL, as determined by the high-resolution crystal structure of the D1.3-HEL complex, were individually truncated to alanine. Only four positions showed a DeltaDeltaG (DeltaGmutant - DeltaGwild-type) of greater than 1.0 kcal/mol, with HEL residue Gln121 proving the most critical for binding (DeltaDeltaG = 2.9 kcal/mol). These residues form a contiguous patch at the periphery of the epitope recognized by D1.3. To understand how potentially disruptive mutations in the antigen are accommodated in the D1.3-HEL interface, we determined the crystal structure to 1.5 A resolution of the complex between D1.3 and HEL mutant Asp18 --> Ala. This mutation results in a DeltaDeltaG of only 0.3 kcal/mol, despite the loss of a hydrogen bond and seven van der Waals contacts to the Asp18 side chain. The crystal structure reveals that three additional water molecules are stably incorporated in the antigen-antibody interface at the site of the mutation. These waters help fill the cavity created by the mutation and form part of a rearranged solvent network linking the two proteins. To further dissect the energetics of specific interactions in the D1.3-HEL interface, double mutant cycles were carried out to measure the coupling of 14 amino acid pairs, 10 of which are in direct contact in the crystal structure. The highest coupling energies, 2.7 and 2.0 kcal/mol, were measured between HEL residue Gln121 and D1.3 residues VLTrp92 and VLTyr32, respectively. The interaction between Gln121 and VLTrp92 consists of three van der Waals contacts, while the interaction of Gln121 with VLTyr32 is mediated by a hydrogen bond. Surprisingly, however, most cycles between interface residues in direct contact in the crystal structure showed no significant coupling. In particular, a number of hydrogen-bonded residue pairs were found to make no net contribution to complex stabilization. We attribute these results to accessibility of the mutation sites to water, such that the mutated residues exchange their interaction with each other to interact with water. This implies that the strength of the protein-protein hydrogen bonds in these particular cases is comparable to that of the protein-water hydrogen bonds they replace. Thus, the simple fact that two residues are in direct contact in a protein-protein interface cannot be taken as evidence that there necessarily exists a productive interaction between them. Rather, the majority of such contacts may be energetically neutral, as in the D1.3-HEL complex.

Amino Acid Substitution↗
PubMed1998

A mutational analysis of the binding of staphylococcal enterotoxins B and C3 to the T cell receptor beta chain and major histocompatibility complex class II.

The three-dimensional structure of the complex between a T cell receptor (TCR) beta chain (mouse Vbeta8.2Jbeta2.1Cbeta1) and the superantigen (SAG) staphylococcal enterotoxin C3 (SEC3) has been recently determined to 3.5 resolution. To evaluate the actual contribution of individual SAG residues to stabilizing the beta-SEC3 complex, as well as to investigate the relationship between the affinity of SAGs for TCR and MHC and their ability to activate T cells, we measured the binding of a set of SEC3 and staphylococcal enterotoxin B (SEB) mutants to soluble recombinant TCR beta chain and to the human MHC class II molecule HLA-DR1. Affinities were determined by sedimentation equilibrium and/or surface plasmon detection, while mitogenic potency was assessed using T cells from rearrangement-deficient TCR transgenic mice. We show that there is a clear and simple relationship between the affinity of SAGs for the TCR and their biological activity: the tighter the binding of a particular mutant of SEC3 or SEB to the TCR beta chain, the greater its ability to stimulate T cells. We also find that there is an interplay between TCR-SAG and SAG-MHC interactions in determining mitogenic potency, such that a small increase in the affinity of a SAG for MHC can overcome a large decrease in the SAG's affinity for the TCR. Finally, we observe that those SEC3 residues that make the greatest energetic contribution to stabilizing the beta-SEC3 complex ("hot spot" residues) are strictly conserved among enterotoxins reactive with mouse Vbeta8.2, thereby providing a basis for understanding why SAGs having other residues at these positions show different Vbeta-binding specificities.

Amino Acid Sequence↗
PubMed1998

Structure of the Vdelta domain of a human gammadelta T-cell antigen receptor.

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.

Amino Acid Sequence↗
PubMed1998

Three-dimensional structure of H-2Dd complexed with an immunodominant peptide from human immunodeficiency virus envelope glycoprotein 120.

The crystal structure of the mouse major histocompatibility complex (MHC) class I molecule H-2Dd with an immunodominant peptide, designated P18-I10 (RGPGRAFVTI), from human immunodeficiency virus envelope glycoprotein 120 was determined at 3.2 A resolution. A novel orientation of the alpha3 domain of Dd relative to the alpha1/alpha2 domains results in significantly fewer contacts between alpha3 and beta2-microglobulin compared with other MHC class I proteins. Four out of ten peptide residues (P2 Gly, P3 Pro, P5 Arg and P10 Ile) are nearly completely buried in the Dd binding groove. This is consistent with previous findings that Dd exploits a four-residue binding motif comprising a glycine at P2, a proline at P3, a positively charged residue at P5, and a C-terminal hydrophobic residue at P9 or P10. The side-chain of P5 Arg is directed toward the floor of the predominantly hydrophobic binding groove where it forms two salt bridges and one hydrogen bond with Dd residue Asp77. The selection of glycine at P2 appears to be due to a narrowing of the B pocket, relative to that of other class I molecules, caused by Arg66 whose side-chain folds down into the binding cleft. Residue P3 Pro of P18-I10 occupies part of pocket D, which in Dd is partially split by a prominent hydrophobic ridge in the floor of the binding groove formed by Trp97 and Trp114. Residues P6 through P9 form a solvent-exposed bulge, with P7 Phe protruding the most from the binding groove and thereby probably constituting a major site of interaction with T cell receptors. A comparison of H-2Dd/P18-I10 with other MHC class I/peptide complexes of known structure provides insights into the possible basis for the specificity of the natural killer cell receptor Ly-49A for several related class I molecules.

Animals↗
PubMed1998

Three-dimensional structure of the complex between a T cell receptor beta chain and the superantigen staphylococcal enterotoxin B.

Superantigens (SAGs) are a class of immunostimulatory proteins of bacterial or viral origin that activate T cells by binding to the V beta domain of the T cell antigen receptor (TCR). The three-dimensional structure of the complex between a TCR beta chain (mouse V beta8.2) and the SAG staphylococcal enterotoxin B (SEB) at 2.4 A resolution reveals why SEB recognizes only certain V beta families, as well as why only certain SAGs bind mouse V beta8.2. Models of the TCR-SEB-peptide/MHC class II complex indicate that V alpha interacts with the MHC beta chain in the TCR-SAG-MHC complex. The extent of the interaction is variable and is largely determined by the geometry of V alpha/V beta domain association. This variability can account for the preferential expression of certain V alpha regions among T cells reactive with SEB.

Animals↗
PubMed1998

Anatomy of an antibody molecule: structure, kinetics, thermodynamics and mutational studies of the antilysozyme antibody D1.3.

Using site-directed mutagenesis, x-ray crystallography, microcalorimetric, equilibrium sedimentation and surface plasmon resonance detection techniques, we have examined the structure of an antibody-antigen complex and the structural and thermodynamic consequences of removing specific hydrogen bonds and van der Waals interactions in the antibody-antigen interface. These observations show that the complex is considerably tolerant, both structurally and thermodynamically, to the truncation of antibody and antigen side chains that form contacts. Alterations in interface solvent structure for two of the mutant complexes appear to compensate for the unfavorable enthalpy changes when antibody-antigen interactions are removed. These changes in solvent structure, along with the increased mobility of side chains near the mutation site, probably contribute to the observed entropy compensation. In concert, data from structural studies, reaction rates, calorimetric measurements and site directed mutations are beginning to detail the nature of antibody-protein antigen interactions.

Alanine↗
PubMed1998

Structure-function studies of T-cell receptor-superantigen interactions.

Superantigens (SAGs) are a class of disease-causing and immunostimulatory proteins of bacterial or viral origin that activate T cells by binding to the V beta domain of the T-cell antigen receptor (TCR). The three-dimensional structure of the complex between a TCR beta chain (mouse V beta 8.2-J beta 2.1-C beta 1) and the SAG staphylococcal enterotoxin C3 (SEC3) has been recently determined. 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 small and large domains of the SAG. A model of the TCR-SAG-peptide/MHC complex constructed from available crystal structures reveals how the SAG acts as a wedge between the TCR and MHC, thereby displacing the antigenic peptide away from the TCR and circumventing the normal mechanism for T-cell activation by peptide/MHC. To evaluate the actual contribution of individual SAG residues to stabilizing the V beta C beta-SEC3 complex, as well as to investigate the relationship between the affinity of SAGs for TCB and MHC and their ability to activate T cells, we measured the binding of a set of SEC3 mutants to a soluble recombinant TCR beta chain and to the human MHC class II molecule HLA-DR1. We show that there is direct correlation between affinity and ability to stimulate T cells, with SAGs having the highest affinity for the TCR being the most biologically active. We also find that there is an interplay between TCR-SAG and SAG-MHC interactions in determining mitogenic potency, such that a small increase in the affinity of a SAG for MHC can overcome a large decrease in the SAG's affinity for the TCR. Finally, we observe that those SEC3 residues that make the greatest energetic contribution to stabilizing the V beta C beta-SEC3 complex are strictly conserved among enterotoxins reactive with mouse V beta 8.2, thereby explaining why SAGs having other residues at these positions show different V beta-binding specificities.

Amino Acid Sequence↗
PubMed1997

Characterization of anti-anti-idiotypic antibodies that bind antigen and an anti-idiotype.

Two mouse monoclonal anti-anti-idiotopic antibodies (anti-anti-Id, Ab3), AF14 and AF52, were prepared by immunizing BALB/c mice with rabbit polyclonal anti-idiotypic antibodies (anti-Id, Ab2) raised against antibody D1.3 (Ab1) specific for the antigen hen egg lysozyme. AF14 and AF52 react with an "internal image" monoclonal mouse anti-Id antibody E5.2 (Ab2), previously raised against D1.3, with affinity constants (1.0 x 10(9) M-1 and 2.4 x 10(7) M-1, respectively) usually observed in secondary responses against protein antigens. They also react with the antigen but with lower affinity (1.8 x 10(6) M-1 and 3.8 x 10(6) M-1). This pattern of affinities for the anti-Id and for the antigen also was displayed by the sera of the immunized mice. The amino acid sequences of AF14 and AF52 are very close to that of D1.3. In particular, the amino acid side chains that contribute to contacts with both antigen and anti-Id are largely conserved in AF14 and AF52 compared with D1.3. Therapeutic immunizations against different pathogenic antigens using anti-Id antibodies have been proposed. Our experiments show that a response to an anti-Id immunogen elicits anti-anti-Id antibodies that are optimized for binding the anti-Id antibodies rather than the antigen.

Amino Acid Sequence↗
PubMed1997

Dual conformations of a T cell receptor V alpha homodimer: implications for variability in V alpha V beta domain association.

The crystal structure of a mutant T cell receptor (TCR) V alpha domain containing a grafted third complementarity-determining region (CDR3) from a different V alpha was determined at 2.3 A resolution by molecular replacement using the wild-type V alpha structure as a search model. Like the wild-type V alpha domain, the mutant crystallized as a homodimer very similar to TCR V alpha V beta and antibody V(L)V(H) heterodimers, with the CDR loops disposed to form part of the antigen-binding site. However, the relative orientation of the two chains in the mutant V alpha homodimer differs from that in the wild-type by a rotation of 14 degrees such that the buried surface area in the dimer interface of the mutant is 140 A2 less than in the wild-type. While the residues forming the interface are essentially the same in the two structures, there are only four pairs of interface hydrogen bonds in the case of the mutant compared with eight for the wild-type. These results suggest that multiple relative orientations of the V alpha and V beta domains of TCRs may be possible, providing a significant contribution to TCR combining site diversity.

Amino Acid Sequence↗
PubMed1997

Analysis of binding interactions in an idiotope-antiidiotope protein-protein complex by double mutant cycles.

The idiotope-antiidiotope complex between the anti-hen egg white lysozyme antibody D1.3 and the anti-D1.3 antibody E5.2 provides a useful model for studying protein-protein interactions. A high-resolution crystal structure of the complex is available [Fields, B. A., Goldbaum, F. A., Ysern, X., Poljak, R.J., & Mariuzza, R. A. (1995) Nature 374, 739-742], and both components are easily produced and manipulated in Escherichia coli. We previously analyzed the relative contributions of individual residues of D1.3 to complex stabilization by site-directed mutagenesis [Dall'Acqua, W., Goldman, E. R., Eisenstein, E., & Mariuzza, R. A. (1996) Biochemistry 35, 9667-9676]. In the current work, we introduced single alanine substitutions in 9 out of 21 positions in the combining site of E5.2 involved in contacts with D1.3 and found that 8 of them play a significant role in ligand binding (delta Gmutant-delta Gwild type > 1.5 kcal/mol). Furthermore, energetically important E5.2 and D1.3 residues tend to be juxtaposed in the crystal structure of the complex. In order to further dissect the energetics of specific interactions in the D1.3-E5.2 interface, double mutant cycles were carried out to measure the coupling of 13 amino acid pairs, 9 of which are in direct contact in the crystal structure. The highest coupling energy (4.3 kcal/mol) was measured for a charged-neutral pair which forms a buried hydrogen bond, while side chains which interact through solvated hydrogen bonds have lower coupling energies (1.3-1.7 kcal/mol), irrespective of whether they involve charged-neutral or neutral-neutral pairs. Interaction energies of similar magnitude (1.3-1.6 kcal/mol) were measured for residues forming only van der Waals contacts. Cycles between distant residues not involved in direct contacts in the crystal structure also showed significant coupling (0.5-1.0 kcal/mol). These weak long-range interactions could be due to rearrangements in solvent or protein structure or to secondary interactions involving other residues.

Antigen-Antibody Complex↗
PubMed1996

Hydrogen bonding and solvent structure in an antigen-antibody interface. Crystal structures and thermodynamic characterization of three Fv mutants complexed with lysozyme.

Using site-directed mutagenesis, X-ray crystallography, and titration calorimetry, we have examined the structural and thermodynamic consequences of removing specific hydrogen bonds in an antigen-antibody interface. Crystal structures of three antibody FvD1.3 mutants, VLTyr50Ser (VLY50S), VHTyr32Ala (VHY32A), and VHTyr101Phe (VHY101F), bound to hen egg white lysozyme (HEL) have been determined at resolutions ranging from 1.85 to 2.10 A. In the wild-type (WT) FvD1.3-HEL complex, the hydroxyl groups of VLTyr50, VHTyr32, and VHTyr101 each form at least one hydrogen bond with the lysozyme antigen. Thermodynamic parameters for antibody-antigen association have been measured using isothermal titration calorimetry, giving equilibrium binding constants Kb (M-1) of 2.6 x 10(7) (VLY50S), 7.0 x 10(7) (VHY32A), and 4.0 x 10(6) (VHY101F). For the WT complex, Kb is 2.7 x 10(8) M-1; thus, the affinities of the mutant Fv fragments for HEL are 10-, 4-, and 70-fold lower than that of the original antibody, respectively. In all three cases entropy compensation results in an affinity loss that would otherwise be larger. Comparison of the three mutant crystal structures with the WT structure demonstrates that the removal of direct antigen-antibody hydrogen bonds results in minimal shifts in the positions of the remaining protein atoms. These observations show that this complex is considerably tolerant, both structurally and thermodynamically, to the truncation of antibody side chains that form hydrogen bonds with the antigen. Alterations in interface solvent structure for two of the mutant complexes (VLY50S and VHY32A) appear to compensate for the unfavorable enthalpy changes when protein-protein interactions are removed. These changes in solvent structure, along with the increased mobility of side chains near the mutation site, probably contribute to the observed entropy compensation. For the VHY101F complex, the nature of the large entropy compensation is not evident from a structural comparison of the WT and mutant complexes. Differences in the local structure and dynamics of the uncomplexed Fv molecules may account for the entropic discrepancy in this case.

Antigen-Antibody Complex↗
PubMed1996

Crystal structure of an Fv-Fv idiotope-anti-idiotope complex at 1.9 A resolution.

Anti-idiotopic antibodies react with unique antigenic features, usually associated with the combining sites, of other antibodies. They may thus mimic specific antigens that react with the same antibodies. The structural basis of this mimicry is analyzed here in detail for an anti-idiotopic antibody that mimics the antigen, hen egg-white lysozyme. The crystal structure of an anti-hen-egg-white lysozyme antibody (D1.3) complexed with an anti-idiotopic antibody (E5.2) has been determined at a nominal resolution of 1.9 A. E5.2 contacts substantially the same residues of D1.3 as lysozyme, thus mimicking its binding to D1.3. The mimicry embodies conservation of hydrogen bonding: six of the 14 protein-protein hydrogen bonds bridging D1.3-E5.2 are structurally equivalent to hydrogen bonds bridging D1.3-lysozyme. The mimicry includes a similar number of van der Waals interactions. The mimicry of E5.2 for lysozyme, however, does not extend to the topology of the non-polar surfaces of E5.2 and lysozyme, which are in contact with D1.3 as revealed by a quantitative analysis of the contacting surface similarities between E5.2 and lysozyme. The structure discussed herein shows that an anti-idiotopic antibody can provide an approximate topological and binding-group mimicry of an external antigen, especially in the case of the hydrophilic surfaces, even though there is no sequence homology between the anti-idiotope and the antigen.

Animals↗
PubMed1996

Crystal structure of a T-cell receptor beta-chain complexed with a superantigen.

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.

Amino Acid Sequence↗
PubMed1996

A mutational analysis of the binding of two different proteins to the same antibody.

The crystal structures of the complexes between the anti-hen egg white lysozyme (HEL) antibody D1.3 and HEL and between D1.3 and the anti-D1.3 antibody E5.2 have shown that D1.3 contacts these two proteins through essentially the same set of combining site residues [Fields, B. A., Goldbaum, F. A., Ysern, X., Poljak, R. J., & Mariuzza, R. A. (1995) Nature 374, 739-742]. To probe the relative contribution of individual residues to complex stabilization, single alanine substitutions were introduced in the combining site of D1.3, and their effects on affinity for HEL and for E5.2 were measured using surface plasmon resonance detection, fluorescence quench titration, or sedimentation equilibrium. The energetics of the binding to HEL are dominated by only 3 of the 13 contact residues tested (delta Gmutant-delta Gwild type > 2.5 kcal/mol): VLW92, VHD100, and VHY101. These form a patch at the center of the interface and are surrounded by residues whose apparent contributions are much less pronounced ( < 1.5 kcal/mol). This contrasts with the interaction of D1.3 with E5.2 in which most the contact residues (11 of 15) were found to play a significant role in ligand binding ( > 1.5 kcal/mol). Furthermore, even though D1.3 contacts HEL and E5.2 in very similar ways, the functionally important residues of D1.3 are different for the two interactions, with only substitutions at D1.3 positions VH100 and VH101 greatly affecting binding to both ligands. Thus, the same protein may recognize different ligands in ways that are structurally similar yet energetically distinct.

Amino Acid Sequence↗
PubMed1996

Crystal structure of the complex of the variable domain of antibody D1.3 and turkey egg white lysozyme: a novel conformational change in antibody CDR-L3 selects for antigen.

The crystal structure of the Fv fragment of the murine monoclonal anti-lysozyme antibody D1.3, complexed with turkey egg-white lysozyme (TEL), is presented. D1.3 (IgG1, kappa) is a secondary response antibody specific for hen egg-white lysozyme (HEL). TEL and HEL are homologous and differ in amino acid sequence in the antibody-antigen interface only at position 121. The side-chain of HEL residue Gln121 makes a pair of hydrogen bonds to main-chain atoms of the antibody light chain. In the D1.3-TEL structure, TEL residue His121 makes only one hydrogen bond with the light chain as a result of 129 degree and 145 degree change in peptide torsion angles for residues Trp92 and Ser93. Probably as a consequence of this conformational change, the D1.3-TEL association occurs at a much slower rate than the D1.3-HEL association. The D1.3-TEL complex is destabilized with respect to the D1.3-HEL interaction by the loss of two hydrogen bonds, exclusively due to the substitution of histidine for glutamine. While antibodies of secondary responses are indeed highly specific for antigen, this work demonstrates that by undergoing subtle conformational change antibodies can still recognize mutated protein antigens, albeit at a cost to affinity.

Animals↗