Kinetic study of phosphorylation-dependent complex formation between the kinase-inducible domain (KID) of CREB and the KIX domain of CBP on a quartz crystal microbalance.

We report quantitative analysis of peptide-peptide interactions on a 27 MHz quartz crystal microbalance (QCM) in aqueous solution. The KID (kinase-inducible domain) of transcription factor CREB (cyclic AMP response element binding protein) is known to interact with the KIX domain of coactivator CBP (CREB binding protein), facilitated by phosphorylation at Ser-133 of the KID. The KIX domain peptide (86 aa) was immobilized on the QCM gold electrode surface by means of a poly(ethylene glycol) spacer. Binding of the KID peptide (46 aa) to the KIX peptide was detected by frequency decreases (mass increases) of the QCM. Both maximum binding amount (Deltammax) and association constants (Ka) obtained from the QCM measurements increased as a result of phosphorylation of Ser-133 of the KID peptide. The Ka values for KIX peptide to the phosphorylated (pKID) and unphosphorylated KID peptides were (93+/-2) x 10(3) and (5+/-1) x 10(3) M(-1), respectively. This difference was explained by the dissociation rate constant (k(-1)) of the pKID being 20 times smaller than that of the KID, while association rate constants (k1) were independent of phosphorylation.