We report the three-dimensional structures, at 1.8-A resolution, ofthe Fv fragment ofthe anti-hen egg white lysozyme antibody D1.3 in its free and antigen-bound forms.These structures reveal a role for solvent molecules in stabilzing 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 salvation of the complex should weaken the hydrophobic effect, and the resulting large number of solvent-mediated hydrogen bonds, in conijunction 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 complementaritydetermining 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.X-ray crystallographic studies of several complexes of antigens with specific antibodies have revealed a high degree of complementarity between their interacting surfaces (reviewed in refs. 1 and 2). Water molecules have been identified at the interfaces of the Fab fragment of antibody D1.3 (Fab D1.3)-hen egg-white lysozyme (HEL) (3) and NC41-neuraminidase (4) complexes on the basis of structure determinations at 2.5-A resolution. Unfortunately, at such resolution, which is about the best which has been so far attained with conventional Fab fragments, the certainty with which ordered water molecules can be located is seriously limited (5). We have now determined the three-dimensional structure, at 1.8-A resolution, of the Fv fragment of monoclonal antibody (mAb) D1.3 (6, 7), Fv D1.3, consisting of only the variable domains ofthe heavy (VH) and light (VL) polypeptide chains and that of its complex with HEL, permitting a more detailed description of an antibody combining site in its free and antigen-bound states. These studies reveal both buried and exposed water molecules linking antigen and antibody and contributing to chemical complementarity between their interacting surfaces.An understanding of how antibodies react with antigens must involve the thermodynamics of the binding interaction. We have therefore experimentally de...

Mitochondria play a key role in apoptosis due to their capacity to release potentially lethal proteins. One of these latent death factors is cytochrome c, which can stimulate the proteolytic activation of caspase zymogens. Another important protein is apoptosis-inducing factor (AIF), a flavoprotein that can stimulate a caspase-independent cell-death pathway required for early embryonic morphogenesis. Here, we report the crystal structure of mouse AIF at 2.0 A. Its active site structure and redox properties suggest that AIF functions as an electron transferase with a mechanism similar to that of the bacterial ferredoxin reductases, its closest evolutionary homologs. However, AIF structurally differs from these proteins in some essential features, including a long insertion in a C-terminal beta-hairpin loop, which may be related to its apoptogenic functions.

Using synthetic Tn (GalNAc-O-Ser/Thr) glycopeptide models and a biosensor based on surface plasmon resonance spectroscopy we have determined that isolectin B4 from Vicia villosa (VVLB4) binds to one Tn determinant whereas the antiTn monoclonal antibodies 83D4 and MLS128 require at least two Tn residues for recognition. When an unglycosylated amino acid is introduced between the Tn residues, both antibodies do not bind. MLS128 affinity was higher on a glycopeptide with three consecutive Tn residues. These results indicate that Tn residues organized in clusters are essential for the binding of these antibodies and indicate a different Tn recognition pattern for VVLB4.z 2000 Federation of European Biochemical Societies.

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.