Fig. 4

Binding mechanisms. a Electrostatic interactions in the binding interface of barnase and barstar. Blue and red sticks indicate positively charged side chains of barnase (cyan) and negatively charged side chains of barstar (orange), respectively. Dashed ellipses show two clusters of interacting residues. b Positively charged side chains (blue sticks) of NCBD (orange molecule) and negatively charged side chains (red sticks) of TAD (cyan molecule). Charged side chains are exposed to the solvent and do not interact except those in one salt bridge indicated by a black arrow. Binding interface is predominantly hydrophobic. c Kinetic scheme of the association via the formation of TC, and proposed free energy surface of TAD/NCBD binding with an arbitrary NCBD concentration at different NaCl concentrations. k₊ and k₋ are the association and dissociation rate coefficients of the formation of TC. k_f is the folding rate of unfolded TAD in TC. k_A = k₊k_f/(k_f + k₋). As [NaCl] is increased, the formation of TC becomes slower (reduced k₊) due to the increased charge screening effect, and TC becomes less stable and dissociates more easily (increased k₋, lowered dissociation barrier) before TAD folds to form a fully bound complex. Both effects decrease k_A. d The transition state configurations (indicated by green O) of barnase (cyan) and barstar (orange) are very similar to that of the bound complex, and therefore, the configurations with unfavorable orientations will not result in association (indicated by red x). Positively and negatively charged side chains in the binding interface are colored in blue and red. On the other hand, the association can be initiated from different parts of NCBD (orange) via non-native interactions between charged residues (i.e., parallel binding pathways^23,24, indicated by green O). Cyan coils represent disordered TAD. Red bars indicate negative charges in TAD and positively charged side chains of NCBD are colored in blue