Crystal Structure of the Complex mAb 17.2 and the C-Terminal Region of Trypanosoma cruzi P2β Protein: Implications in Cross-Reactivity

Patients with Chronic Chagas' Heart Disease possess high levels of antibodies against the carboxyl-terminal end of the ribosomal P2ß protein of Trypanosoma cruzi (TcP2ß). These antibodies, as well as the murine monoclonal antibody (mAb) 17.2, recognize the last 13 amino acids of TcP2ß (called the R13 epitope: EEEDDDMGFGLFD) and are able to cross-react with, and stimulate, the ß1 adrenergic receptor (ß1-AR). Indeed, the mAb 17.2 was able to specifically detect human β1-AR, stably transfected into HEK cells, by flow cytometry and to induce repolarisation abnormalities and first degree atrioventricular conduction block after passive transfer to naïve mice. To study the structural basis of this cross-reactivity, we determined the crystal structure of the Fab region of the mAb 17.2 alone at 2.31 Å resolution and in complex with the R13 peptide at 1.89 Å resolution. We identified as key contact residues on R13 peptide Glu3, Asp6 and Phe9 as was previously shown by alanine scanning. Additionally, we generated a model of human β1-AR to elucidate the interaction with anti-R13 antibodies. These data provide an understanding of the molecular basis of cross-reactive antibodies induced by chronic infection with Trypanosoma cruzi

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.

International audienceThe 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

Crystallization and preliminary X-ray diffraction study of the bacterially expressed Fv from the monoclonal anti-lysozyme antibody D1.3 and of its complex with the antigen, lysozyme

The associated heavy (VH) and light (VL) chain variable domains (FV) of the monoclonal anti-lysozyme antibody D1.3, secreted from Escherichia coli, have been crystallized in their antigen-bound and free forms. FvD1.3 gives tetragonal crystals, space group P41212 (or P43212), with a = 90·6 Å, c = 56·4 Å. The FvD1.3-lysozyme complex crystallizes in space group C2, with a = 129·2 Å, b = 60·8 Å, c = 56·9 Å and β = 119·3°. The crystals contain one molecule of Fv or of the Fv-lysozyme complex in their asymmetric units and diffract X-rays to high resolution, making them suitable for X-ray crystallographic studies.</p

Polar contacts in the model between 17.2 and β1-AR.

<p>MC: Main Chain; SC: Side Chain.</p

Crystallographic data and refinement statistics.

<p>Crystallographic data and refinement statistics.</p

Model of the interaction of Fab 17.2 with the human β1-AR.

<p>A. Superposition of the second extracellular loop (2ECL) of the human β1-AR model (violet) and the region 2–5 of the epitope (light green). B. Complex of the Fab 17.2 (light green) with the human β1-AR (violet) inserted in a membrane model (grey). C. Zoom of the paratope region of 17.2 interacting with the second extracellular loop. The main contact points are illustrated as dotted yellow lines. Heavy chain CDRs are coloured in red and light chain CDRs in blue.</p

Functional activity of the mAb 17.2.

<p>A. Representative histogram showing the HEK and HEK-β1 cells labelled with mAb 17.2 followed by a Cy3-conjugated goat anti-mouse IgG. B. Results are expressed as means ± SD (n = 3). Inset: Binding of mAb 17.2 to HEK-β1 cells in the presence of R13 peptide. ** <i>p</i><0.01; *** <i>p</i><0.001. C. Passive transfer of mAb 17.2 to naïve mice. Repolarisation abnormalities (a) and first degree AV conduction block (b) are indicated by arrows. bpm: beats per minute.</p

Structure of the Fab 17.2.

<p>A. Apo Fab 17.2 structure (molecule 1). Heavy chain CDRs are coloured in red and light chain CDRs in blue. B. Superposition of molecules 1, VH-VL region of the two crystals asymmetric units. C. Superposition of molecules 2, VH-VL region of the two crystals asymmetric units. Apo Fab 17.2 (grey), Fab 17.2-R13 molecule 1 (green) and molecule 2 (magenta).</p

Structure of the Fab17.2 – R13 complex.

<p>A. Superposition of the apo Fab 17.2 and R13 complex structures (grey and green respectively). VH and VL contact residues are indicated. B. Main water molecules present on the antigen binding site of Fab 17.2 apo. C. Superposition of water molecules present in the apo Fab 17.2 that are replaced by the peptide in the Fab 17.2 R13 complex. D. Superposition of R13 peptides from molecules 1 (green) and 2 (magenta). E. Structure of the Fab 17.2-R13 complex (molecule 1). All hydrogen bonds between mAb 17.2 and peptide R13 are illustrated as dotted yellow lines. The π-stacking interaction between VL Tyr101 and the epitope Phe9 is also indicated. Heavy chain CDRs are coloured in red and light chain CDRs in blue the peptide is coloured in light green.</p