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1 Department of Molecular Biology,
2 Department of Chemistry, and
3 The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California 92037, USA
4 Howard Hughes Medical Institute and The Salk Institute for Biological Studies, La Jolla, California 92037, USA
Reprint requests to: Peter E. Wright, Department of Molecular Biology, MB-2, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA; e-mail: wright{at}scripps.edu; fax: (858) 7849822.
Among the most common interaction motifs between nuclear proteins is the recognition of one or more amphipathic helices. In an effort to determine principles behind this recognition, we have investigated the interaction between the p160 coactivator protein ACTR and the ACTR-binding domain of the CREB-binding protein, CBP. The two proteins use relatively small portions of their primary sequences to form a single synergistically folded domain consisting of six intertwined 
-helices, three from each protein. Neither of the component polypeptides forms a cooperatively folded domain in isolation. However, a considerable amount of residual secondary structure remains in the isolated CBP domain according to CD spectroscopy. Chemical denaturation, differential scanning calorimetry, and ANS binding experiments demonstrate that the isolated CBP domain is not entirely unfolded but forms a helical state with the characteristics of a molten globule. Mutations probing the functional and energetic significance of a buried intermolecular Arg–Asp salt bridge in the interface of the protein complex suggest that these residues are tuned for functional discrimination and not strictly for binding affinity or stability. These results suggest a mechanism for formation of the complex where the unfolded ACTR domain interacts with the partly folded CBP domain in a rapid and specific manner to form the final stable complex.
Keywords: CABD of ACTR; NCBD of CBP; folding upon binding; molten globule; intrinsically unstructured protein
Abbreviations: ACTR, activator for thyroid hormone and retinoid receptors • ANS, 1-anilinonapthalene-8-sulfonate • CBP, CREB binding protein • CD, circular dichroism • DSC, differential scanning calorimetry • HAT, histone acetyltransferase • ITC, isothermal titration calorimetry • LBD, ligand binding domain • NHR, nuclear hormone receptor • NMR, nuclear magnetic resonance • SRC1, steroid receptor coactivator 1 • TIF2, transcription intermediary factor 2 • NCBD, nuclear coactivator binding domain of CBP • CABD, coactivator binding domain of ACTR
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