Appropriate integration of cellular signals requires a delicate balance of ligand–target binding affinities. Increasing the level of residual structure in intrinsically disordered proteins (IDPs), which are overrepresented in these cellular processes, has been shown previously to enhance binding affinities and alter cellular function. Conserved proline residues are commonly found flanking regions of IDPs that become helical upon interacting with a partner protein. Here, we mutate these helix-flanking prolines in p53 and MLL and find opposite effects on binding affinity upon an increase in free IDP helicity. In both cases, changes in affinity were due to alterations in dissociation, not association, rate constants, which is inconsistent with conformational selection mechanisms. We conclude that, contrary to previous suggestions, helix-flanking prolines do not regulate affinity by modulating the rate of complex formation. Instead, they influence binding affinities by controlling the lifetime of the bound complex.

The relationship between helical stability and binding affinity was examined for the intrinsically disordered transactivation domain of the myeloblastosis oncoprotein, c-Myb, and its ordered binding partner, KIX. A series of c-Myb mutants was designed to either increase or decrease helical stability without changing the binding interface with KIX. This included a complimentary series of A, G, P, and V mutants at three non-interacting sites. We were able to use the glycine mutants as a reference state and show a strong correlation between binding affinity and helical stability. The intrinsic helicity of c-Myb is 21%, and helicity values of the mutants ranged from 8% to 28%. The c-Myb helix is divided into two conformationally distinct segments. The N-terminal segment, from K291-L301, has an average helicity greater than 60% and the C-terminal segment, from S304-L315, has an average helicity less than 10%. We observed different effects on binding when these two segments were mutated. Mutants in the N-terminal segment that increased helicity had no effect on the binding affinity to KIX, while helix destabilizing glycine and proline mutants reduced binding affinity by more than 1 kcal/mol. Mutants that either increased or decreased helical stability in the C-terminal segment had almost no effect on binding. However, several of the mutants reveal the presence of multiple conformations accessible in the bound state based on changes in enthalpy and linkage analysis of binding free energies. These results may explain the high level of sequence identity (>90%), even at non-interacting sites, for c-Myb homologues.