Structural Evidence for Entropic Contribution of Salt Bridge Formation to a Protein Antigen-Antibody Interaction

Abstract

A structural and thermodynamic study of the entropic contribution of salt bridge formation to the interaction between hen egg white lysozyme (HEL) and the variable domain fragment (Fv) of anti-HEL antibody, HyHEL-10, was carried out. Three Fv mutants (HD32A, HD96A, and HD32AD96A) were prepared, and the interactions between the mutant Fvs and HEL were investigated. Crystallography revealed that the overall structures of these mutant complexes were almost identical to that of wildtype Fv. Little structural changes were observed in the HD32AD96A mutant-HEL complex, and two water molecules were introduced into the mutation site, indicating that the two water molecules structurally compensated for the complete removal of the salt bridges. This result suggests that the entropic contribution of the salt bridge originates from dehydration. In the singly mutated complexes, one water molecule was also introduced into the mutated site, bridging the antigen-antibody interface. However, a local structural difference was observed in the HD32A Fv-HEL complex, and conformational changes occurred due to changes in the relative orientation of the heavy chain to the light chain upon complexation in HD96A Fv-HEL complexes. The reduced affinity of these single mutants for the antigen originates from the increase in entropy loss, indicating that these structural changes also introduced an increase in entropy loss. These results suggest that salt bridge formation makes an entropic contribution to the protein antigen-antibody interaction through reduction of entropy loss due to dehydration and structural changes.The ability of proteins to bind to one another in a highly specific manner is an important feature of biological phenomena, and the mechanism of antibody specificity and affinity toward target proteins has been studied as a model of proteinprotein interactions (1). X-ray crystal structures of antibodies complexed with proteinaceous antigens (2-6) suggest that the strict specificity of an antibody originates from a high degree of complementarity between the antigen-combining sites and the antigen. The complementarity is composed of two factors: 1) apolar surfaces interacting by van der Waals contact, where the surfaces of the antibody and antigen often fit together to exclude most hydrated water molecules from the interface and favorable close van der Waals contacts are formed; and 2) polar surfaces interacting by hydrogen bond and salt bridge formation, where the precise positioning of atoms allows chargecharge interactions (i.e. salt bridge formation and hydrogen bond formation).To elucidate the roles of the noncovalent forces involved in antigen-antibody complementary association, both structural and thermodynamic information are crucial (7). X-ray crystal analysis can clarify the structural aspects for the complementarity of the interactions (2-5), and titration calorimetry can provide useful information for the quantitative assessment of the contribution of residues to the interaction (8 -10). Thus, the combination of these...

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