Conserved Helix-Flanking Prolines Modulate Intrinsically Disordered Protein:Target Affinity by Altering the Lifetime of the Bound Complex.

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

Disorder Levels of c-Myb Transactivation Domain Regulate its Binding Affinity to the KIX Domain of CREB Binding Protein

Intrinsically disordered proteins (IDPs) do not form stable tertiary structures like their ordered partners. They exist as heterogeneous ensembles that fluctuate over a time scale. Intrinsically disordered regions and proteins are found across different phyla and exert crucial biological functions. They exhibit transient secondary structures in their free state and become folded upon binding to their protein partners via a mechanism called coupled folding and binding. Some IDPs form alpha helices when bound to their protein partners. We observed a set of cancer associated IDPs where the helical binding segments of IDPs are flanked by prolines on both the sides. Helix-breaking prolines are frequently found in IDPs flanking the binding segment and are evolutionarily conserved across phyla. Two studies have shown that helix flanking prolines modulate the function of IDPs by regulating the levels of disorder in their free state and in turn regulating the binding affinities to their partners. We aimed to study if this is a common phenomenon in IDPs that exhibit similar pattern in the conservation of helix flanking prolines. We chose to test the hypothesis in c-Myb-KIX : IDP-target system in which the disordered protein exhibits high residual helicity levels in its free state. c-Myb is a hematopoietic regulator that plays a crucial role in cancer by binding to the KIX domain of CBP. Studying the functional regulation of c-Myb by modulating the disorder levels in c-Myb and in IDPs in general provides a better understanding of the way IDPs function and can be used in therapeutic strategies as IDPs are known to be involved in regulating various cellular processes and diseases. To study the effect of conserved helix flanking prolines on the residual helicity levels of c-Myb and its binding affinity to the KIX domain of CBP, we mutated the prolines to alanines. Mutating prolines to alanines increased the helicity levels of c-Myb in its free state. This small increase in the helicity levels of a highly helical c-Myb showed almost no effect on the binding affinity between cMyb and KIX. We hypothesized that there is a helical threshold for coupled folding and binding beyond which helicity levels of the free state IDP have no effect on its binding to their ordered protein partner. To test this hypothesis, we mutated solvent exposed amino acid residues in c-Myb that reduce its overall helicity and studied its effect on the binding affinity between c-Myb and KIX. Over a broad range of reduction in helicity levels of the free state did not show an effect on the binding affinity but beyond a certain level, decrease in helicity levels showed pronounced effects on the binding affinity between c-Myb and KIX

Uncoupling the Folding and Binding of an Intrinsically Disordered Protein

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 (\u3e 90%), even at non-interacting sites, for c-Myb homologues

Uncoupling the Folding and Binding of an Intrinsically Disordered Protein

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 (\u3e 90%), even at non-interacting sites, for c-Myb homologues