Thermodynamic analysis of an antibody functional epitope.
We have probed the relative contribution of polar and nonpolar interactions to antibody-antigen interaction by measuring the effect of single amino acid substitutions in an humanized anti-p185HER2 antibody (hu4D5-5) on the thermodynamics of antigen binding. First we mapped the functional epitope by complete alanine-scan mutagenesis of the antibody complementarity-determining region (CDR). Four residues, H91 in VL and R50, W95, and Y100a in VH, make large contributions to the free energy of binding (delta delta G > 3 kcal mol-1) and have delta delta G > delta delta H. These residues are clustered in a shallow pocket on the antibody surface in the X-ray structure determined for hu4D5 Fv. The majority of other CDR residues make less energetically important contributions (delta delta G < 1 kcal mol-1) to binding but have delta delta H > delta delta G, suggesting that the wild-type side chain does contact antigen but the loss in entropy, perhaps through restriction of side-chain conformational freedom, offsets the favorable enthalpic term. Effects of Phe and Ala substitutions on the delta G and delta Cp of antigen binding indicate that the favorable contribution of antibody tyrosine residues to binding results primarily from burial of the aromatic ring in the interface with antigen. Burial of the phenyl ring has a favorable delta H at 25 degrees C but at least for one site (VL-Y92) is opposed by delta S. This latter feature is inconsistent with the thermodynamics predicted for the hydrophobic effect based on hydrocarbon-transfer experiments.(ABSTRACT TRUNCATED AT 250 WORDS)
Kelley
R F
RF
Protein Engineering Department, Genentech, Inc., South San Francisco, California 94080.
O'Connell
M P
MP
D000906:Antibodies / Q000235:genetics / Q000276:immunology; D000937:Antigen-Antibody Reactions; D001666:Binding Sites, Antibody; D000939:Epitopes / Q000737:chemistry*; D007700:Kinetics; D016296:Mutagenesis; D011487:Protein Conformation; D013816:Thermodynamics
D000906:Antibodies; D000939:Epitopes