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Although antibodies are highly specific, cross-reactions are frequently observed. To understand the molecular basis of this phenomenon, we studied the anti-hen egg lysozyme (HEL) monoclonal antibody (mAb) D11.15, which cross-reacts with several avian lysozymes, in some cases with a higher affinity (heteroclitic binding) than for HEL. We have determined the crystal structure of the Fv fragment of D11.15 complexed with pheasant egg lysozyme (PHL). In addition, we have determined the structure of PHL, Guinea fowl egg lysozyme, and Japanese quail egg lysozyme. Differences in the affinity of D11.15 for the lysozymes appear to result from sequence substitutions in these antigens at the interface with the antibody. More generally, cross-reactivity is seen to require a stereochemically permissive environment for the variant antigen residues at the antibody-antigen interface.

The crystal structure of the complex between the cross-reacting antigen Guinea fowl lysozyme and the Fab from monoclonal antibody F9.13.7, raised against hen egg lysozyme, has been determined by x-ray diffraction to 3-A resolution. The antibody interacts with exposed residues of an alpha-helix and surrounding loops adjacent to the lysozyme active site cleft. The epitope of lysozyme bound by antibody F9.13.7 overlaps almost completely with that bound by antibody HyHEL10; the same 12 residues of the antigen interact with the two antibodies. The antibodies, however, have different combining sites with no sequence homology at any of their complementarity-determining regions and show a dissimilar pattern of cross-reactivity with heterologous antigens. Side chain mobility of epitope residues contributes to confer steric and electrostatic complementarity to differently shaped combining sites, allowing functional mimicry to occur. The capacity of two antibodies that have different fine specificities to bind the same area of the antigen emphasizes the operational character of the definition of an antigenic determinant. This example demonstrates that degenerate binding of the same structural motif does not require the existence of sequence homology or other chemical similarities between the different binding sites.

Crystallographic analysis indicated that Clostridium thermocellum endoglucanase CelD contained three Ca(2+)-binding sites, termed A, B, and C, and one Zn(2+)-binding site. The protein contributed five, six, and three of the coordinating oxygen atoms present at sites A, B, and C, respectively. Proteins altered by mutation in site A (CelDD246A), B (CelDD361A), or C (CelDD523A) were compared with wild type CelD. The Ca(2+)-binding isotherm of wild type CelD was compatible with two high affinity sites (Ka = 2 x 10(6) M-1) and one low affinity site (Ka < 10(5) M-1). The Ca(2+)-binding isotherms of the mutated proteins showed that sites A and B were the two high affinity sites and that site C was the low affinity site. Atomic absorption spectrometry confirmed the presence of one tightly bound Zn2+ atom per CelD molecule. The inactivation rate of CelD at 75 degrees C was decreased 1.9-fold upon increasing the Ca2+ concentration from 2 x 10(-5) to 10(-3) M. The Km of CelD was decreased 1.8-fold upon increasing the Ca2+ concentration from 5 x 10(-6) to 10(-4) M. Over similar ranges of concentration, Ca2+ did not affect the thermostability nor the kinetic properties of CelDD523A. These findings suggest that Ca2+ binding to site C stabilizes the active conformation of CelD in agreement with the close vicinity of site C to the catalytic center.

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.

Adult bovine aortic tissue was treated with 6 M guanidinium chloride in the presence of proteinase inhibitors to obtain an extract that was essentially devoid of collagenous components and appeared homogeneous by electron microscopy. When this extract was dispersed by sonication it was found to be a very potent inducer of human platelet aggregation. This interaction required the presence of von Willebrand factor and of its receptor (glycoprotein Ib) on platelet membrane. This was demonstrated by the fact that the aggregation of normal blood platelets resuspended in plasmas deficient in von Willebrand factor was significantly diminished as compared to aggregation in control plasma. Moreover, this aggregation was inhibited by a monoclonal antibody, IgG AN51, to platelet glycoprotein Ib. These studies provide direct biochemical evidence for the existence of a thrombogenic constituent of the vessel wall that is noncollagenous and von Willebrand factor-dependent.