A model of transcriptional activator-coactivator recognition is provided by the mammalian CREB activation domain and the KIX domain of coactivator CBP. The CREB kinase-inducible activation domain (pKID, 60 residues) is disordered in solution and undergoes an alpha-helical folding transition on binding to CBP [Radhakrishan, I., Perez-Alvarado, G. C., Parker, D., Dyson, H. J., Montminy, M. R., and Wright, P. E. (1997) Cell 91, 741-752]. Binding requires phosphorylation of a conserved serine (RPpSYR) in pKID associated in vivo with the biological activation of CREB signaling pathways. The CBP-bound structure of CREB contains two alpha-helices (designated alphaA and alphaB) flanking the phosphoserine; the bound structure is stabilized by specific interactions with CBP. Here, the nascent structure of an unbound pKID domain is characterized by multidimensional NMR spectroscopy. The solubility of the phosphopeptide (46 residues) was enhanced by truncation of N- and C-terminal residues not involved in pKID-CBP interactions. Although disordered under physiologic conditions, the pKID fragment and its unphosphorylated parent peptide exhibit partial folding at low temperatures. One recognition helix (alphaA) is well-defined at 4 degreesC, whereas the other (alphaB) is disordered but inducible in 40% trifluoroethanol (TFE). Such nascent structure is independent of serine phosphorylation and correlates with the relative extent of engagement of the two alpha-helices in the pKID-KIX complex; whereas alphaA occupies a peripheral binding site with few intermolecular contacts, the TFE-inducible alphaB motif is deeply engaged in a hydrophobic groove. Our results support the use of TFE as an empirical probe of hidden structural propensities and define a correspondence between induced fit and the nascent structure of peptide fragments.