Stimulus-specific interaction between activator-coactivator cognates revealed with a novel complex-specific antiserum

A number of second messenger pathways propagate inductive signals via protein-protein interactions that are phosphorylation-dependent. The second messenger, cAMP, for example, promotes cellular gene expression via the protein kinase A-mediated phosphorylation of cAMP-response element-binding protein (CREB) at Ser(133), and this modification in turn stimulates the association of CREB with the co-activator, CREB-binding protein (CBP). The solution structure of the CREB.CBP complex, using relevant interaction domains, kinase inducible domain and kinase-induced domain interacting domain, referred to as KID and KIX, respectively, shows that KID undergoes a coil to helix transition, upon binding to KIX, that stabilizes complex formation. Whether such changes occur in the context of the full-length CREB and CBP proteins, however, is unclear. Here we characterize a novel antiserum that specifically binds to the CREB. CBP complex but to neither protein individually. Epitope mapping experiments demonstrate that the CREB.CBP antiserum detects residues in KID that undergo a conformational change upon binding to KIX. The ability of this antiserum to recognize full-length CREB.CBP complexes in a phospho-(Ser(133))-dependent manner demonstrates that the structural transition observed with the isolated KID domain also occurs in the context of the full-length CREB protein. To our knowledge, this is the first report documenting formation of endogenous cellular protein-protein complexes in situ.     

Phosphorylation-induced transient intrinsic structure in the kinase-inducible domain of CREB facilitates its recognition by the KIX domain of CBP

Phosphorylation at Ser-133 of the kinase inducible domain of CREB (KID) triggers its binding to the KIX domain of CBP via a concomitant coil-to-helix transition. The exact role of this key event is still puzzling: it does not switch between disordered and ordered states, nor its direct interactions fully account for selectivity. Hence, we reasoned that phosphorylation may shift the conformational preferences of KID towards a binding-competent state. To this end we investigated the intrinsic structural properties of the unbound KID in phosphorylated and unphosphorylated forms by simulated annealing and molecular dynamics simulations. Although helical populations show subtle differences, phosphorylation reduces the flexibility of the turn segment connecting the two helices in the complexed structure and induces a transient structural element that corresponds to its bound conformation. It is stabilized by the pSer-133-Arg-131 interaction, which is absent from the unphosphorylated KID. Diminishing this coupling decreases the 3.1 kcal/mol contribution of pSer-133 to the binding free energy (DeltaGbind) of the phosphorylated KID to KIX by 1.1 kcal/mol, as computed in reference to Ser-133. In a binding competent form of the S133E KID mutant, the contribution of Glu-133 to DeltaGbind is by 1.5 kcal/mol smaller than that of pSer, suggesting that altered structural properties due to pSer --> Glu replacement impair the binding affinity. Thus, we propose that phoshorylation contributes to selectivity not merely by the direct interactions of the phosphate group with KIX, but also by promoting the formation of a transient structural element in the highly conserved turn segment.