On the dynamic nature of the transition state for protein-protein association as determined by double-mutant cycle analysis and simulation

The process of protein-protein association starts with their random collision, which may develop into an encounter complex followed by a transition state and final complex formation. Here we aim to experimentally characterize the nature of the transition state of protein-protein association for three different protein-protein interactions; Barnase-Barstar, TEM1-BLIP and IFNalpha2-IFNAR2, and use the data to model the transition state structures. To model the transition state, we determined inter-protein distance-constraints of the activated complex by using double mutant cycles (DMC) assuming that interacting residues are spatially close. Significant DeltaDeltaG(double dagger)(int) values were obtained only between residues on Barnase and Barstar. However, introducing specific mutations that optimize the charge complementarity between BLIP and TEM1 resulted in the introduction of significant DeltaDeltaG(double dagger)(int) values also between residues of these two proteins. While electrostatic interactions make major contributions towards stabilizing the transition state, we show two examples where steric hindrance exerts an effect on the transition state as well. To model the transition-state structures from the experimental DeltaDeltaG(double dagger)(int) values, we introduced a method for structure perturbation, searching for those inter-protein orientations that best support the experimental DeltaDeltaG(double dagger)(int) values. Two types of transition states were found, specific as observed for Barnase-Barstar and the electrostatically optimized TEM1-BLIP mutants, and diffusive as shown for wild-type TEM1-BLIP and IFNalpha2-IFNAR2. The specific transition states are characterized by defined inter-protein orientations, which cannot be modeled for the diffusive transition states. Mutations introduced through rational design can change the transition state from diffusive to specific. Together, these data provide a structural view of the mechanism allowing rates of association to differ by five orders of magnitude between different protein complexes.