Binding Mode and Transcriptional Activation Potential of High Affinity Ligands for the CBP KIX Domain

Abstract

We recently described a pair of ligands, PPKID4(P) (4(P)) and PPKID6(U) (6(U)), which present the alpha-helical functional epitope found on helix B of the CREB KID activation domain (KID(P)) on a pancreatic fold protein scaffold. 4(P) and 6(U) bind the natural target of KID(P), the KIX domain of the coactivator CBP, with equilibrium dissociation constants between 515 nM and 1.5 microM and compete effectively with KID(P) for binding to CBP KIX (KIX). Here we present a detailed investigation of the binding mode, orientation, and transcriptional activation potential of 4(P) and 6(U). Equilibrium binding experiments using a panel of well-characterized KIX variants support a model in which 4(P) binds KIX in a manner that closely resembles that of KID(P) but 6(U) binds an overlapping, yet distinct region of the protein. Equilibrium binding experiments using a judiciously chosen panel of 4(P) variants containing alanine or sarcosine substitutions along the putative alpha- or PPII helix of 4(P) support a model in which 4(P) folds into a pancreatic fold structure upon binding to KIX. Transcriptional activation assays performed in HEK293 cells using GAL4 DNA-binding domain fusion proteins indicate that 4(P) functions as a potent activator of p300/CBP-dependent transcription. Notably, 6(U) is a less potent transcriptional activator in this context than 4(P)despite the similarity of their affinities for CBP KIX. This final result suggests that thermodynamic affinity is an important, although not exclusive, criterion controlling the level of KIX-dependent transcriptional activation.

Kinetic modulation of a disordered protein domain by phosphorylation

“…The fact that the glutamate mutation cannot sustain long-lived states and disrupts 25 the binding is consistent with this proposed mechanism of modulation. Previous studies have shown that pSer133 contributes to binding via specific intermolecular contacts, and a loss of affinity is seen when mutating specific residues in KIX 25,28,52 . The exact atomistic mechanism and whether these contacts are also sufficient alone for binding is less clear.…”

Scaffolds for Blocking Protein-Protein Interactions

“…Subsequent second-and third-generation library synthesis resulted in a small grafted protein displaying a K i value of 35 nM for hDM2 binding. Further studies included investigations into miniature proteins designed to mimic the kinase-inducible activation domain (KID) of the transcription factor CREB with the KIX domain of the transcriptional coactivator protein CPB, [15] as well as that of the cAMP-dependent protein kinase recognition epitope found in the heat-stable protein kinase inhibitor protein [16].…”

Trapping Moving Targets with Small Molecules

“…Previous inhibition studies primarily focused on short peptides that competitively associated with the shallow hydrophobic KID binding cleft of KIX (37). However, NMR screening of 762 preselected small molecule ligands demonstrated that protein-protein interactions of this complex can be regulated by trapping an inactive conformer (38).…”