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

SUNDBERG EJ, URRUTIA M, BRADEN BC, ISERN J, TSUCHIYA D, FIELDS BA, MALCHIODI EL, TORMO J, SCHWARZ FP, MARIUZZA RA.

BIOCHEMISTRY

AMER CHEMICAL SOC

Lugar: Washington; Año: 2000 vol. 39 p. 15375 - 15387

0006-2960

Antigen-antibody complexes provide useful models for analyzing the thermodynamicsof protein-protein association reactions. We have employed site-directedmutagenesis, X-ray crystallography, and isothermal titration calorimetry toinvestigate the role of hydrophobic interactions in stabilizing the complexbetween the Fv fragment of the anti-hen egg white lysozyme (HEL) antibody D1.3and HEL. Crystal structures of six FvD1.3-HEL mutant complexes in which aninterface tryptophan residue (V(L)W92) has been replaced by residues with smallerside chains (alanine, serine, valine, aspartate, histidine, and phenylalanine)were determined to resolutions between 1.75 and 2.00 A. In the wild-type complex,V(L)W92 occupies a large hydrophobic pocket on the surface of HEL and constitutesan energetic "hot spot" for antigen binding. The losses in apolar buried surface area in the mutant complexes, relative to wild-type, range from 25 (V(L)F92) to115 A(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 apeptide flip. The affinities of the mutant Fv fragments for HEL are 10-100-foldlower than that of the original antibody. Formation of all six mutant complexesis marked by a decrease in binding enthalpy that exceeds the decrease in binding free energy, such that the loss in enthalpy is partly offset by a compensatinggain in entropy. No correlation was observed between decreases in apolar, polar, or aggregate (sum of the apolar and polar) buried surface area in the V(L)92mutant series and changes in the enthalpy of formation. Conversely, there existlinear correlations between losses of apolar buried surface and decreases inbinding free energy (R(2) = 0.937) as well as increases in the solvent portion ofthe entropy of binding (R(2) = 0.909). The correlation between binding freeenergy and apolar buried surface area corresponds to 21 cal mol(-1) A(-2) (1 cal = 4.185 J) for the effective 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 energeticfactor in the formation of this protein complex. Our estimate of the hydrophobic contribution to binding at site V(L)92 in the D1.3-HEL interface is consistentwith values for the hydrophobic effect derived from classical hydrocarbonsolubility models. We also show how residue V(L)W92 can contribute significantly less to stabilization when buried in a more polar pocket, illustrating thedependence of the hydrophobic effect on local environment at different sites in aprotein-protein interface.