Contribution to stability and folding of a buried polar residue at the CARM1 methylation site of the KIX domain of CBP.

Abstract

The transcriptional coactivator and acetyltransferase CREB Binding Protein (CBP) is comprised of several autonomously folded and functionally independent domains. The KIX domain mediates interactions between CBP and numerous transcriptional activators. The folded region of KIX has all the structural features of a globular protein, including three alpha-helices, two short 3(10) helices, and a well-packed hydrophobic core. KIX contains a buried cation-pi interaction between the positively charged guanidinium group of Arg 600 and the aromatic ring of Tyr 640. Arg 600 is a site for regulatory methylation by CARM1/PRMT4, which negates the CREB-binding function of the KIX domain. The role of the Arg 600-Tyr 640 buried polar interaction in specifying and stabilizing the structure of KIX was investigated by comparing the folding of wild-type KIX with the single point mutants Y640F and R600M. The Y640F mutant disrupts a hydrogen bond involving the Tyr 640 OH and the backbone of V595 but still allows for the cation-pi interaction while the R600M mutant disrupts the cation-pi interaction. Both wild type KIX and Y640F exhibit properties expected of native like, globular proteins such as a single oligomerization state (monomer), cooperative thermal and urea-induced unfolding transitions, and a well-packed core. In contrast, the R600M mutant has properties reminiscent of a molten globule state, including a tendency to aggregate, noncooperative thermal unfolding transition, and a loosely packed core. Thus, the buried cation-pi interaction is critical for specifying the unique cooperatively folded structure of KIX.