Bound water molecules and conformational stabilization help mediate an antigen-antibody association.

Abstract

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...

Kinetic epitope mapping of the chicken lysozyme.HyHEL‐10 fab complex: Delineation of docking trajectories

“…Intermolecular contacts in protein-protein interfaces include hydrogen bonds, electrostatic, and van der Waals interactions. Bound solvent molecules are released (Bhat et al, 1994), and decreases in the conformational entropies of interacting side chains ensue upon association. The relative contributions of each of these factors is likely to be system dependent.…”

Water–protein interactions from high–resolution protein crystallography

“…Many physico-chemical techniques have been applied to visualize hydration structures, and particular contributions have been made using X-ray (Watenpaugh et al 1978;Blake et al 1983;Saenger 1987;Baker et al 1988;Teeter 1991) and neutron crystallography (Finner-Moore et al 1992;Kossiakoff et al 1992). Crystallographic studies provide structural evidence about the predominant contribution of water molecules inside proteins in respect of their biological functions, such as catalytic efficiencies (Zhang et al 1999), molecular recognition (Bhat et al 1994), stabilization of the higher-order structures of Figure 1. A schematic but realistic illustration of proteins around the cell surface.…”

Structural mechanism for affinity maturation of an anti-lysozyme antibody

“…Structural studies, as well as epitope mapping (9)(10)(11), have contributed a wealth of information regarding the structural aspects of the anti-lysozyme response. The three-dimensional structures of eight complexes between HEL and the Fab or Fv fragments of murine anti-HEL antibodies have been reported, identifying several important features of antibody͞antigen interactions (12)(13)(14)(15)(16)(17)(18)(19)(20)(21). The specificity of binding is determined almost exclusively by the structure of the complementarity-determining regions (CDRs) of the V H and V L domains.…”