Macromolecular interactions are central to cellular regulation and biological function, and antibody-antigen complexes are often used as a paradigms for molecular recognition. Protein-protein interactions are studied utilizing monoclonal antibodies (mAbs) specific for hen egg white lysozyme, a protein which has long served as a prototype for investigating the specificity of immune recognition. mAbs HH10, HH26, HH63, and HH8 recognize highly coincident epitopes and share over 90% sequence homology, but they differ significantly in their specificity properties. Differing cross-reactivities make the 4 Abs appear to have different sizes of functional epitopes as defined by alanine scanning performed using lysozyme which has been expressed and mutated in yeast. Differences in overall sensitivity of the mAbs to mutations in the antigen epitope reflect differences among them in the relative stabilities of their association and dissociation rate constants, to antigenic variation. HH8, the most cross-reactive, has fast on rates and slow off rates, and both rates are quite insensitive to antigenic variation. In contrast, both rates of HH26, the Ab whose sequence is most similar to that of the germ line genes, are sensitive to antigenic variation: mutations in the antigen both slow on rates and increase off rates. The structurally defined HH10 is intermediate in antigen sensitivity, with stable on rates but variable off rates. There are significant correlations between the intramolecular contacts, hydrophobicity, CDR flexibility, kinetics, and specificity of these mAbs. Recently, a high resolution structure of the Fab63-HEL complex has been refined by my postdoctoral fellow Y. Li in the laboratory of our collaborator, Dr. R. Mariuzza, and a high resolution of the Fab26-HEL complex is in progress. Comparison of these structures with that of Fab10-HEL will allow interpretation of the molecular basis of the biophysical differences among the complexes.The antibodies have been expressed as Fab in E. coli, and chain and domain swaps made. Results from the chimeric antibodies support the hypothesis that the stable on rate of HH8 correlates with H2, which is much more hydrophobic than H2 of the other antibodies, while differences among off-rates stabilities correlate with only a few residues in the L chains. Results with site-directed mutations support the hypothesis that specificity and affinity are significantly modulated by intramolecular salt link networks involving noncontact residues. We hypothesize that these salt link networks are largely responsible for conformational flexibility differences among the antibodies, which in turn modulate specificity and affinity. Thus, important structural differences among the 3 mAbs which determine the functional differences involve indirect, long-range effects by noncontact residues. It has been proposed that this type of conformational modulation may play an important role in antibody affinity maturation.Using BIAcore surface plasmon resonance technology, we found that the real-time association kinetics of Fabs specific for hen egg-white lysozyme did not conform to a 1:1 Langmuir association model. Binding of all 4 monovalent Fabs to lysozyme is best described by a 2-step kinetic model, the most simplified scheme that quantitatively describes an encounter followed by a docking/conformational rearrangement: A+B<==>[AB]*<==>AB. The first (encounter) step is defined by the rate constants k+1 and k-1, and the equilibrium constant Ka1=(k+1/ k-1), and the docking step by Ka2=(k+2/ k-2). The net affinity KA=Ka1(1+Ka2. Estimates of encounter rate constants k+1 and k-1 for H26 association with HEL are in good agreement with rate constants determined by atomic force microscopy (collaborative project with Dr. Peter Hinterdorfer). We have defined a new parameter, T50, which specifies the biological half-life of each encounter complex and the time at which the encounter and final complexes are of equimolar concentration. The observed T50 is a function of analyte concentration and the encounter and docking rate constants. Simulations showed that when the ligand is saturated at high analyte concentrations, T50 reaches a minimum value, T50(min), which can be used to compare antigen-antibody complexes. This parameter gives a comparative measure of how quickly a given complex forms a tight association, and may be a more biologically relevant measure of activity than affinity. Analysis of binding kinetics using the 2-step model has allowed the following insight into the dynamics of antibody-antigen association: (1) Values of T50 are unique for each antibody-antigen complex. The encounter complexes of unmutated Fabs with HEL have biological half lives (T50) of 5-10 mins, while the decreased affinities with mutant antigens often reflect significantly slower docking with longer T50 of an hour or longer. (2) The same antigenic mutation affects different steps in the associations of the three Abs. (3) Faster net off rates accompanying decreased antibody affinity for mutant antigens may reflect significantly slower docking (slower k+2) with longer T50 but not faster rate constants k- 1 and k-2. (4) Degree of antibody cross-reactivity with mutant antigens correlates with free energy changes in the docking step, consistent with our hypothesis that cross- reactivity reflects conformational flexibility of the combining site. Our results suggest that the free energy barriers to conformational rearrangement are highest in noncross-reactive antibodies and lowest in cross-reactive antibodies, especially in complexes with mutant anitigens. (5) Our results predict that H8 would be the most and H26 the least entropically driven, a result that has been confirmed with calorimetric measurements (in collaboration with Dr. R. Willson). (6) Temperature differentially affects the encounter and docking steps, altering T50 and the distribution of free energy change between these two steps. The lower affinity observed at higher temperatures is attributable to a temperature-dependent decrease in docking rate.The results of these studies are directly applicable to rational design of antibodies with predesigned specificity and dynamics for diagnostic and therapeutic applications. They also should be applicable to other protein-protein receptor-ligand interactions.
