Estimation of the hydrophobic effect in an antigen-antibody protein-protein interface

Antigen-antibody complexes provide useful models for analyzing the thermody namics of protein-protein association reactions. We have employed site-dire cted mutagenesis, X-ray crystallography, and isothermal titration calorimet ry to investigate the role of hydrophobic interactions in stabilizing the c omplex between the Fv fragment of the anti-hen egg white lysozyme (HEL) ant ibody D1.3 and HEL. Crystal structures of six FvD1.3-HEL mutant complexes i n which an interface tryptophan residue (V(L)W92) has been replaced by resi dues with smaller side chains (alanine, serine, valine, aspartate, histidin e, and phenylalanine) were determined to resolutions between 1.75 and 2.00 Angstrom. In the wild-type complex, V(L)W92 occupies a large hydrophobic po cket on the surface of HEL and constitutes an energetic "hot spot" for anti gen binding. The losses in apolar buried surface area in the mutant complex es, relative to wild-type, range from 25 (V(L)F92) to 115 Angstrom (2) (V(L )A92), with no significant shifts in the positions of protein atoms at the mutation site for any of the complexes except V(L)A92, where there is a pep tide flip. The affinities of the mutant Fv fragments for HEL are 10-100-fol d lower than that of the original antibody. Formation of all six mutant com plexes is marked by a decrease in binding enthalpy that exceeds the decreas e in binding free energy, such that the loss in enthalpy is partly offset b y a compensating gain in entropy. No correlation was observed between decre ases in apolar, polar, or aggregate (sum of the apolar and polar) buried su rface area in the V(L)92 mutant series and changes in the enthalpy of forma tion. Conversely, there exist linear correlations between losses of apolar buried surface and decreases in binding free energy (R-2 = 0.937) as well a s increases in the solvent portion of the entropy of binding (R-2 = 0.909). The correlation between binding free energy and apolar buried surface area corresponds to 21 cal mol(-1) Angstrom (-2) (1 cal = 4.185 J) for the effe ctive hydrophobicity at the V(L)92 mutation site. Furthermore, the slope of the line defined by the correlation between changes in binding free energy and solvent entropy approaches unity, demonstrating that the exclusion of solvent from the binding interface is the predominant energetic factor in t he formation of this protein complex. Our estimate of the hydrophobic contr ibution to binding at site V(L)92 in the D1.3-HEL interface is consistent w ith values for the hydrophobic effect derived from classical hydrocarbon so lubility models. We also show how residue V(L)W92 can contribute significan tly less to stabilization when buried in a more polar pocket, illustrating the dependence of the hydrophobic effect on local environment at different sites in a protein-protein interface.