Packing, specificity, and mutability at the binding interface between the p160 coactivator and CREB-binding protein

Authors

  • Stephen J. Demarest,

    1. Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA
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  • Songpon Deechongkit,

    1. Department of Chemistry, The Scripps Research Institute, La Jolla, California 92037, USA
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  • H. Jane Dyson,

    1. Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA
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  • Ronald M. Evans,

    1. Howard Hughes Medical Institute and The Salk Institute for Biological Studies, La Jolla, California 92037, USA
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  • Peter E. Wright

    Corresponding author
    1. Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA
    2. The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California 92037, USA
    • Department of Molecular Biology, MB-2, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA; fax: (858) 7849822.
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Abstract

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 α-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.

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