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 (HEL), a protein which has long served as a prototype for investigating the specificity of immune recognition. Four structurally and functionally related mAbs, H8, H10, H26, and H63, recognize highly coincident epitopes and share over 90% sequence homology, but they differ significantly in their fine specificity properties. Our recent finding, using BIAcore surface plasmon resonance technology (SPR), that the real-time association kinetics of all 4 Fabs specific for HEL are best described by a 2-step kinetic model, has completely changed our view of the association process in these complexes, and led us to develop totally new protocols for kinetic analyses that are significantly different from those conventionally used in the literature. Although bimolecular association has for over 20 years generally been considered to occur in 2 steps, in practice, our results are the first to experimentally confirm a 2-step binding mechanism for antibody-protein complexes. We have applied transition state theory to describe the free energy changes of 2-step binding of bimolecular complexes, an advance in the theoretical foundation of protein dynamics. Our application of the model to the 2-step (3-state) binding of antibody complexes reveals experimentally determined free energy change profiles signicantly different from those proposed in the literature, which assume that the encounter complex is energetically unfavorable. For all wild type and mutant complexes examined to date, the totla activation energy required for docking (approximately 20 kcal/mol) is about twice that required to from the encounter complex (9-10kcal/mol). In the complexes of H8 and H10 with HEL, the maximum free energy level attained is during the encounter transition state, thud thus the encounter step is the rate limiting step of the association. We have applied van't Hoff analysis to the 2-stepkinetics of these complexes, in order to calculate binding and activation thermodynamics of the separate steps as well as of net association. We find that the thermodynamics of the encounter and docking steps are significantly different from each other. The thermodynamics of encounter are consistent with a hydrophobically driven process. The energetics of docking are consistent with formation of noncovalent bonds and conformational rearrangements. There is entropy-enthalpy compensation within each step, and also between the steps. These are the properties that have long been predicted for the 2 steps of the association process, but protein-protein complexes that display these properties are rarely reported, probably because the 2 steps usually cannot be separated. Preliminary results suggest that antigenic mutations alter the thermodynamic balance between the steps. These results support the hypotheses that (i) docking includes a conformational change, (ii) hydrophobic interactions predominate energetics of one of the antibodies, while those of the other 3 reflect a range of more polar and electrostatic interactions The net changes agree with calorimetry measurements on the same complexes by our collaborator R.C. Willson, U Houston, and provide new insight into the mechanisms underlying frequently observed inconsistencies between theromdonamics derived from calorimetry and kinetics. Calorimetry is apparently biased towards the docking step while van't Hoff thermodynamics calculated from BIAcore kinetics using widely prevailing methods are biased towards the encounter step. In addition, recent experiments with osmolytes have allowed estimations of movements of water molecules during association; the encounter step is accompanied by water exclusion, after the formation of the transition state, which is consistent with our results which indicate the encounter step is entropically driven. In contrast, docking is accompanied by water uptake, both before and after the formation of the transtion state. These results are consistent with a step which is enthalpically driven, as indicated by our results. It is becoming apparent that there may be fundamental thermodynamic differences in receptor binding by agonist and antagonists. Understanding thermodynamics of binding can provide information on the nature of transition states and underlying reaction mechanisms. A detailed understanding of the thermodynamics of these structurally and functionally characterized complexes is therefore of theorectical and practical interest. In collaboration with R. Mariuzza, CARB, we have determined several new x-ray structures, including, those of H26 and of the recombinant antibody H8L10 complexed with HEL. In addition, the complexes of H63 Fab complexed with several site directed mutants. These structures have provided new insight into the role of electrostatics and flexibility in complex formation, and together with the thermodynamic data provide a new view of antibody affinity maturation.Our computational studies support the hypothesis that cross-reactivity requires conformational flexibility of the Ab combining site, largely modulated by intramolecular salt links and networks; while rigidly preorganized binding sites produce more specific binding. A major obstacle to the de novo structure-based design of ligands and of receptors with predefined specificity, including Abs, is an incomplete understanding of the roles of receptor mobility and conformational changes that accompany ligand binding. Our results 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.
