Three-dimensional structure and thermodynamics of antigen binding by anti-lysozyme antibodies.
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Animals↗
Biomedical subjects
R A Mariuzza
Publications and source records attributed to R A Mariuzza.
Preferential V beta gene usage and lack of junctional sequence conservation among human T cell receptors specific for a tetanus toxin-derived peptide: evidence for a dominant role of a germline-encoded V region in antigen/major histocompatibility complex recognition.
To investigate the structural and genetic basis of the T cell response to defined peptide/major histocompatibility (MHC) class II complexes in humans, we established a large panel of T cell clones (61) from donors of different HLA-DR haplotypes and reactive with a tetanus toxin-derived peptide (tt830-844) recognized in association with most DR molecules (universal peptide). By using a bacterial enterotoxin-based proliferation assay and cDNA sequencing, we found preferential use of a particular V beta region gene segment, V beta 2.1, in three of the individuals studied (64%, n = 58), irrespective of whether the peptide was presented by the DR6wcI, DR4w4, or DRw11.1 and DRw11.2 alleles, demonstrating that shared MHC class II antigens are not required for shared V beta gene use by T cell receptors (TCRs) specific for this peptide. V alpha gene use was more heterogeneous, with at least seven different V alpha segments derived from five distinct families encoding alpha chains able to pair with V beta 2.1 chains to form a tt830-844/DR-specific binding site. Several cases were found of clones restricted to different DR alleles that expressed identical V beta and (or very closely related) V alpha gene segments and that differed only in their junctional sequences. Thus, changes in the putative complementary determining region 3 (CDR3) of the TCR may, in certain cases, alter MHC specificity and maintain peptide reactivity. Finally, in contrast to what has been observed in other defined peptide/MHC systems, a striking heterogeneity was found in the junctional regions of both alpha and beta chains, even for TCRs with identical V alpha and/or V beta gene segments and the same restriction. Among 14 anti-tt830-844 clones using the V beta 2.1 gene segment, 14 unique V beta-D-J beta junctions were found, with no evident conservation in length and/or amino acid composition. One interpretation for this apparent lack of coselection of specific junctional sequences in the context of a common V element, V beta 2.1, is that this V region plays a dominant role in the recognition of the tt830-844/DR complex.
Amino Acid Sequence↗
Crystallographic refinement of the three-dimensional structure of the FabD1.3-lysozyme complex at 2.5-A resolution.
The three-dimensional crystal structure of the complex between the Fab from the monoclonal anti-lysozyme antibody D1.3 and the antigen, hen egg white lysozyme, has been refined by crystallographic techniques using x-ray intensity data to 2.5-A resolution. The antibody contacts the antigen with residues from all its complementarity determining regions. Antigen residues 18-27 and 117-125 form a discontinuous antigenic determinant making hydrogen bonds and van der Waals interactions with the antibody. Water molecules at or near the antigen-antibody interface mediate some contacts between antigen and antibody. The fine specificity of antibody D1.3, which does not bind (K alpha less than 10(5) M-1) avian lysozymes where Gln121 in the amino acid sequence is occupied by His, can be explained on the basis of the refined model.
Amino Acid Sequence↗
Lysine 271 in the transmembrane domain of the T-cell antigen receptor beta chain is necessary for its assembly with the CD3 complex but not for alpha/beta dimerization.
The T-cell antigen receptor (TcR) complex present on most T-cells is formed by a clone-specific disulfide-linked alpha/beta heterodimer noncovalently associated to the CD3 complex, the latter composed of five invariant polypeptides: gamma, delta, epsilon, zeta/zeta, or zeta/eta. The presence of conserved, oppositely charged, amino acids in the predicted transmembrane domains of all the subunits of the TcR.CD3 complex suggests that these residues may have a critical function in the assembly and/or stabilization of the complex. In order to analyze the role of the transmembrane-charged amino acids in the association and cell surface expression of the TcR.CD3 complex, we have carried out site-directed mutagenesis of Lys271 in the transmembrane domain of the TcR beta chain and analyzed the capacity of the altered chain to assemble in a TcR beta-negative T-cell line. Here we show that substitution of this positively charged residue by alanine or glutamine does not prevent cytoplasmic association of alpha and beta chains to form disulfide-linked heterodimers, but does abolish formation of an alpha/beta.CD3 complex and, consequently, its expression on the cell surface.
Antigens, CD↗
Three-dimensional structure determination of an anti-2-phenyloxazolone antibody: the role of somatic mutation and heavy/light chain pairing in the maturation of an immune response.
The three-dimensional structure of the Fab fragment of an anti-2-phenyloxazolone monoclonal antibody (NQ10/12.5) in its native and complexed forms has been determined at 2.8 and 3.0 A resolution, respectively. Identification of hapten-contacting residues has allowed us to evaluate the contribution of individual somatic point mutations to maturation of the immune response. In particular, amino acid residues 34 and 36 of the light chain, which are frequently mutated in antibodies with increased affinity for 2-phenyloxazolone, are shown to interact directly with the hapten. We propose that the strict maintenance of certain amino acid sequences at the potentially highly variable VL-JL and VH-D-JH junctions observed among anti-2-phenyloxazolone antibodies is due largely to structural constraints related to antigen recognition. Finally, the three-dimensional model of NQ10/12.5, which uses the typical light chain of primary response anti-2-phenyloxazolone antibodies but a different heavy chain, allows an understanding of how, by preserving key contact residues, a given heavy chain may be replaced by another, apparently unrelated one, without loss of hapten binding activity and why the V kappa Ox1 germline gene is so frequently selected amongst the other known members of this family.
Amino Acid Sequence↗
A human TCR-Ig chimeric protein used to generate a TCR alpha chain variable region-specific mAb.
In a recent report, a construction containing the alpha chain-variable region (V alpha) coding sequence of a cDNA clone derived from a diphtheria toxoid-specific human T cell (P28), fused to a human immunoglobulin kappa light chain constant region (Ck), was used stably to transfect a murine myeloma cell. In the present study, these transfected cells were employed as an immunogen to raise a mAb, termed 1C5V alpha, specific both for the V alpha Ck chimeric protein secreted by the transfectant and the P28 T cell antigen receptor-V alpha region. mAb 1C5V alpha specifically immunoprecipitates the V alpha Ck protein as a family of 32-35 kDa bands present in the 35S-methionine-labeled culture supernatant from the transfected cells. It specifically binds clone P28. Surface molecules recognized by mAb 1C5V alpha are physically linked to the CD3 molecules since cell treatment with either 1C5V alpha or anti-CD3 mAbs caused the simultaneous down-regulation of the CD3/TCR molecular complex. This link is further supported by immunoprecipitation experiments. Thus, both the 1C5V alpha and the anti-CD3 mAbs precipitate the 16-28 kDa CD3 molecules and the disulfide-linked form of P28 TCR from 125I-labeled P28 T cells. Studies performed in order to define whether a stimulus directly acting on the TCR-V alpha region may trigger the intracellular events observed during human T cell activation showed that (a) mAb 1C5V alpha efficiently triggers the phospholipase C transduction pathway revealed by an accelerated phosphoinositides turn-over and an increased production of phosphorylated derivatives of inositol phosphates; (b) mAb 1C5V alpha induces an up-regulation of IL2R mRNA, accompanied by a slight increase of IL2 and IFN alpha mRNA transcripts evidently amplified in the presence of PMA; (c) soluble mAb 1C5V alpha is strongly mitogenic together with PMA. These results provide the first evidence for the structural authenticity of a secreted water-soluble chimeric form of the variable region of a human TCR alpha chain. They further demonstrate that such chimeric proteins may be valuable tool to further dissect the various functional structure of the human TCR.
Antibodies, Monoclonal↗
Three-dimensional structure and antigen binding specificity of antibodies.
A number of specific Fab and Fv fragments and their complexes with antigens (avian lysozymes), haptens, and anti-idiotopic Fabs have been studied by immunochemical and crystallographic techniques. Antigen and antibody interact through closely complementary contacting surfaces, without major conformational changes. An idiotopic determinant of a monoclonal antibody is shown to include parts of most of its complementarity determining regions. The specificity of antigen recognition resides in the close complementarity of the antigenic determinant with the antibody combining site.
Animals↗
Studies of structure and specificity of some antigen-antibody complexes.
By using X-ray diffraction and immunochemical techniques, we have exploited the use of monoclonal antibodies raised against hen egg lysozyme (HEL) to study systematically those factors responsible for the high specificity of antigen-antibody interactions. HEL was chosen for our investigations because its three-dimensional structure and immunochemistry have been well characterized and because naturally occurring sequence variants from different avian species are readily available to test the fine specificity of the antibodies. The X-ray crystal structure of a complex formed between HEL and the Fab D1.3 shows a large complementary surface with close interatomic contacts between antigen and antibody. Thus single amino acid sequence changes in heterologous antigens give antigen-antibody association constants that are several orders of magnitude smaller than that of the homologous antigen. For example, a substitution of His for Glu at position 121 in the antigen is sufficient to diminish significantly the binding between D1.3 and the variant lysozyme. The conformation of HEL when complexed to D1.3 shows no significant difference from that seen in the free molecule, and immunobinding studies with other anti-HEL antibodies suggest that this observation may be generally true for the system of monoclonal antibodies that we have studied.
Antibody Specificity↗
Secretion of a homodimeric V alpha C kappa T-cell receptor-immunoglobulin chimeric protein.
Transfection into lymphoid cells of a chimeric T-cell receptor-immunoglobulin gene has been used to generate a secreted water-soluble form of the variable (V) domain of a human T-cell receptor alpha chain for use in structural (i.e. x-ray crystallographic) studies. The chimeric protein consists of the V alpha region of the T-cell receptor of a diphtheria toxoid-specific human T-cell clone fused to a human immunoglobulin kappa light chain constant (C) region. It is efficiently secreted by myeloma cells as a noncovalent homodimer of 65-kDa molecular mass in the absence of either immunoglobulin heavy or light chain. The V alpha C kappa protein is extensively glycosylated, and its secretion is glycosylation-dependent. Chimeric genes containing the V beta region of this particular T-cell receptor linked to immunoglobulin C kappa or C gamma 2 regions are expressed intracellularly, but the products, although glycosylated, are not secreted, nor do they assemble with the V alpha C kappa protein. This suggests that the chimeric beta chain-immunoglobulin proteins are incorrectly folded and/or processed due either to the design of the gene fusions themselves or to the absence of vital T-cell-specific accessory molecules in the myeloma host.
Amino Acid Sequence↗
The structural basis of antigen-antibody recognition.
We have reviewed here the three-dimensional structure of an antigen-Fab complex as determined by X-ray crystallographic studies. The antigen is hen egg-white lysozyme (HEL), a protein whose three-dimensional structure and antigenic properties are well known. The Fab was prepared from a murine monoclonal anti-HEL antibody, IgGl,kappa, obtained by cell-hybridization techniques. The equilibrium association constant for the complex is 4.5 X 10(7) mol-1. The complex was crystallized and its three-dimensional structure was determined at 6-A and 2.8-A resolution. A three-dimensional model of the structure was built based on electron-density maps and the amino-acid sequence [determined from the nucleotide sequence of cDNA clones (M. Verhoeyen, C. Berek, J.M. Jarvis, G. Winter, in preparation)]. The three-dimensional structure of the complex shows that 17 antibody residues make close contacts (less than or equal to 4 A) with 16 antigen residues. Fifteen of the contacting antibody residues belong to the six complementarity-determining regions of the light chain (6 residues) and of the heavy chain (9 residues). The remaining two are located in regions of constant or nearly constant sequence ["framework" regions]. The 16 contacting lysozyme residues form a discontinuous, topographical determinant, since they are widely separated in the linear amino-acid sequence but are brought to relative spatial proximity by the three-dimensional folding of the polypeptide chain. The contacting surfaces are relatively flat, with protruding side chains of antigen and antibody penetrating each other over an area with maximum dimensions of 30 X 20 A. As in several other systems of protein-protein interactions, the contacts are chemically characterized as van der Waals interactions and hydrogen bonds. Detailed analysis of the interactions reveals that the antibody's recognition of the antigen is finely specific and is affected by antigenic variation (as observed in lysozymes from other avian species). The quaternary structure of the complexed Fab is elongated, with the axes of the variable (VH + VL) and constant (CH1 + CL) domains making an angle close to 180 degrees. Comparison of the three-dimensional structure of the complexed lysozyme with that of native lysozyme showed no significant conformational change at the current resolution (2.8 A). Comparison of the Fab moiety of the complex with other Fabs of known three-dimensional structure suggested that upon complexing no conformational change takes place in the tertiary structure of Fab either.(ABSTRACT TRUNCATED AT 400 WORDS)
Antibodies↗
Three-dimensional structure of an antigen-antibody complex at 2.8 A resolution.
The 2.8 A resolution three-dimensional structure of a complex between an antigen (lysozyme) and the Fab fragment from a monoclonal antibody against lysozyme has been determined and refined by x-ray crystallographic techniques. No conformational changes can be observed in the tertiary structure of lysozyme compared with that determined in native crystalline forms. The quaternary structure of Fab is that of an extended conformation. The antibody combining site is a rather flat surface with protuberances and depressions formed by its amino acid side chains. The antigen-antibody interface is tightly packed, with 16 lysozyme and 17 antibody residues making close contacts. The antigen contacting residues belong to two stretches of the lysozyme polypeptide chain: residues 18 to 27 and 116 to 129. All the complementarity-determining regions and two residues outside hypervariable positions of the antibody make contact with the antigen. Most of these contacts (10 residues out of 17) are made by the heavy chain, and in particular by its third complementarity-determining region. Antigen variability and antibody specificity and affinity are discussed on the basis of the determined structure.
Animals↗
The predicted structure of immunoglobulin D1.3 and its comparison with the crystal structure.
Predictions of the structures of the antigen-binding domains of an antibody, recorded before its experimental structure determination and tested subsequently, were based on comparative analysis of known antibody structures or on conformational energy calculations. The framework, the relative positions of the hypervariable regions, and the folds of four of the hypervariable loops were predicted correctly. This portion includes all residues in contact with the antigen, in this case hen egg white lysozyme, implying that the main chain conformation of the antibody combining site does not change upon ligation. The conformations of three residues in each of the other two hypervariable loops are different in the predicted models and the experimental structure.
Amino Acid Sequence↗
Preliminary crystallographic study of the Fab fragments of two monoclonal anti-2-phenyloxazolone antibodies.
We report on the preparation, crystallization, and preliminary x-ray crystallographic study of the Fab fragments of two monoclonal anti-2-phenyloxazolone antibodies obtained from the secondary response to this hapten. The Fab fragment from one of these (NQ10/12.5) has been crystallized from polyethylene glycol 8000 solutions in a form suitable for high-resolution x-ray crystallographic studies. These crystals are monoclinic, space group C2, with a = 129.2 A, b = 79.4 A, c = 57.7 A, beta = 96.2 degrees, and one Fab/asymmetric unit. Determination of the three-dimensional structure of Fab NQ10/12.5 should help clarify the role of somatic mutation in the maturation of an immune response. This antibody and an anti-lysozyme antibody also under study apparently use the same germ-line encoded VK and a similar VH gene, respectively, as the idiotypic anti-oxazolone antibodies characteristic of the primary response. A comparative study of the two structures should shed light on the role of the pairing of heavy and light chains in the antigen-binding function of antibodies.
Animals↗
Crystallization of the fab fragments of monoclonal anti-p-azophenylarsonate antibodies and their complexes with haptens.
We report on the preparation, crystallization, and preliminary x-ray crystallographic study of Fab fragments from monoclonal anti-p-azophenylarsonate antibodies. Several crystalline forms were obtained with the Fab fragment from the R19.9 monoclonal antibody as well as with the complex between the hapten p-aminobenzenearsonic acid and Fab R19.9. The crystals of this hapten-Fab complex are similar to but not always isomorphous with the native Fab crystals. All the native and complex crystals were obtained using polyethylene glycol 6000 as crystallizing agent. Some of these crystalline forms diffract to a 2-A resolution or beyond and are suitable for high resolution x-ray diffraction analysis. A possible interpretation of hapten binding to crystalline Fab fragments from R19.9 and from the R9.3 monoclonal anti-p-azophenylarsonate antibody, implying conformational changes, is discussed.
Animals↗
Preliminary crystallographic study of the complex between the Fab fragment of a monoclonal anti-lysozyme antibody and its antigen.
Preliminary crystallographic data are given for the complex between the Fab fragment of a monoclonal anti-lysozyme antibody and its antigen. This crystalline complex was found by screening a number of Fab-lysozyme complexes prepared from monoclonal anti-lysozyme antibodies produced by hybrids of BALB/c immune spleen cells with a non-secreting mouse hybrid myeloma line. The complex crystallizes in the monoclinic space group P21 with a = 55.5 (+/- 0.1) A, b = 143.5 (+/- 0.3) A, c = 49.1 (+/- 0.1) A, beta = 120 degrees 20' (+/- 10'). X-ray photographs show reflections extending to a resolution of 2.7 A. The crystals are suitable for high-resolution X-ray diffraction studies.
Antibodies, Monoclonal↗
Preliminary crystallographic study of the Fab fragment of a monoclonal anti-phenylarsonate antibody possessing a major idiotypic specificity.
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Animals↗
Crystals of the human heavy chain disease protein Riv and human Fc fragment are isomorphous: further evidence for conformational flexibility in the hinge region of immunoglobulins.
Protein Riv is a human gamma 1 heavy chain disease immunoglobulin variant with a deletion of the entire VH and CH1 domains and consisting of most of the hinge region plus the CH2 and CH3 domains. Crystals of this protein are orthorhombic, belonging to the space group P2(1)2(1)2(1), with a = 80.1 A, b = 145.5 A, c = 50.1 A. These crystals are shown to be isomorphous with crystals of a human Fc fragment, indicating that the hinge region and the initial part of the CH2 domain of protein Riv do not assume a unique conformation in the crystalline state.
Crystallization↗
Preliminary crystallographic study of the Fab fragment of a monoclonal anti-phenylarsonate antibody.
Preliminary crystallographic data are given for the Fab fragment of a monoclonal anti-p-phenylarsonate antibody. This crystalline Fab fragment was found by screening a number of monoclonal anti-arsonate antibodies obtained from hybrids of A/J immune spleen cells with a non-secreting mouse myeloma line. The protein crystallizes in the monoclinic space group P21 with a = 86.2 +/- 0.1 A, b = 80.4 +/- 0.2 A, c = 75.8 +/- 0.1 A, beta = 90.3 +/- 0.1 degrees. Precession photographs show X-ray reflections extending to a resolution of 3 A.
Antibodies, Monoclonal↗