We recently described a pair of ligands, PPKID4(P) (4(P)) and PPKID6(U) (6(U)), which present the a-helical functional epitope found on helix B of the CREB KID activation domain (KIDP) on a pancreatic fold protein scaffold. 4(P) and 6(U) bind the natural target of KIDP, the KIX domain of the coactivator CBP, with equilibrium dissociation constants between 515 nM and 1.5 mu M and compete effectively with KIDP for binding to CBP KIX (KIX).(1) 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 KIDP 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 4P 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.