Abstract
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.
Highlights
Disordered proteins (IDPs) or intrinsically disordered regions of proteins (IDRs) lack a well-defined three-dimensional fold and exist as ensembles of conformations with variable levels of transient structure.[1]
Using stopped-flow fluorescence, we investigated the kinetic basis for the increase in affinity of p53 for MDM2, upon mutation of helix-flanking prolines to alanine (PtoA)
If the increased level of residual structure increased the proportion of binding competent p53, i.e., a conformational selection mechanism, the proline to alanine mutation (PtoA) mutations would be expected to increase the association rate constant, yet despite increasing the residual helicity within the region that becomes helical upon binding by 2.5-fold,[3] an only 1.23 ± 0.06-fold increase in kon (Figure 2A) was observed for the proline 27 to alanine (P27A) mutant
Summary
Disordered proteins (IDPs) or intrinsically disordered regions of proteins (IDRs) lack a well-defined three-dimensional fold and exist as ensembles of conformations with variable levels of transient structure.[1]. We have previously shown that this is the case for the disordered transactivation domain of p53, where mutation of conserved helix-flanking prolines increases the peak residual helicity of the free state by approximately 40%.3. When the proline 27 to alanine (P27A) mutation is present, this increase in residual helicity is accompanied by a 10-fold increase in affinity for its ordered binding partner, MDM2. It has been suggested that observing a concomitant enhancement in IDR residual helicity and IDR:target partner protein affinity, will lead to an increase in the proportion of binding competent species within the conformational ensemble and, an increase in the rate of complex formation.[4,8]. We demonstrate that the principal effect is on complex lifetime, rather than rate of formation, but that conserved helix-flanking prolines have more subtlety in the way that they can control affinity than a simple model might predict
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