Effect of Transient Helicity of cMyb TAD on its Binding Affinity to the Kix Domain of CBP/p300

Abstract

Intrinsically disordered proteins (IDPs) are involved in various protein‐protein interactions and they often experience a coupled folding and binding reaction when they bind to their protein partners. Many IDPs, like the cMyb trans activation domain (cMyb TAD), exhibit transient helical structures in solution that resemble their bound structures. We hypothesize that the levels of transient helicity in the free IDP will affect the binding affinity to their protein partners in a predictable way. Previous studies have shown that increasing the transient helicity of intrinsically disordered p53 tumor suppressor increases the binding affinity to MDM2 and had negative effects on cellular function. We aim to study the effect of changing cMyb TAD transient helicity on its binding affinity to the kix domain of CBP/p300.cMyb is a hematopoietic regulator which is known to play a role in tumorigenesis. Unlike p53 TAD, cMyb TAD has a high residual helicity in solution in the absence of binding partners. Prolines that flank the N and C‐terminus of this helical region were mutated to alanines to determine whether there was a change in helicity that would affect the binding of Kix. Upon mutation, the increase in helicity had almost no effect on the binding affinity of the cMyb‐Kix complex. Further, mutations that reduce the helicity of cMyb‐TAD were designed to test the effect of reducing transient helicity on the binding affinity to Kix. To achieve this goal, conservative mutations of the solvent exposed amino acids in cMyb were made. We are currently measuring the transient helicity and binding affinity of these mutants with the long‐range goal of identifying a general relationship between changes in transient helicity and binding affinity of IDPs.Support or Funding InformationThis research was supported by funding to GWD by National Institutes of Health (2R01CA14124406‐A1 and 1R01GM115556‐01A1). This work was also supported by funding to JC by Wellcome trust (WT 095195MA) and by a Biotechnology and Biological Sciences Research Council (BBSRC) studentship to MDC.