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Packing, specificity, and mutability at the binding interface between the p160 coactivator and CREB-binding protein
Stephen J Demarest1, Songpon Deechongkit, H Jane Dyson
1Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Protein Science : a Publication of the Protein Society
|December 24, 2003
Summary
Nuclear protein interactions often involve amphipathic helices. This study reveals how ACTR and CREB-binding protein (CBP) form a stable complex, highlighting specific binding mechanisms for nuclear coactivator recognition.
Area of Science:
- Molecular Biology
- Protein Interactions
- Biochemistry
Background:
- Nuclear protein interactions frequently utilize amphipathic helix motifs.
- Understanding these recognition principles is crucial for deciphering cellular signaling pathways.
Purpose of the Study:
- Investigate the interaction between the p160 coactivator protein ACTR and the ACTR-binding domain of CREB-binding protein (CBP).
- Determine the principles governing the recognition and formation of this specific protein complex.
Main Methods:
- Circular dichroism (CD) spectroscopy to assess secondary structure.
- Chemical denaturation and differential scanning calorimetry to analyze protein stability.
- Anilinonaphthalene sulfonate (ANS) binding assays to probe molten globule states.
- Site-directed mutagenesis to investigate the role of specific residues (Arg-Asp salt bridge).
Main Results:
- ACTR and CBP form a stable, synergistically folded domain composed of six intertwined alpha-helices (three from each protein).
- Isolated CBP domain exhibits residual secondary structure, characteristic of a molten globule state.
- Specific intermolecular salt bridges (Arg-Asp) are optimized for functional discrimination rather than solely binding affinity or stability.
Conclusions:
- The formation of the ACTR-CBP complex involves the rapid and specific interaction of an unfolded ACTR domain with a partially folded CBP molten globule state.
- This mechanism facilitates the formation of a stable, functional protein complex, offering insights into nuclear coactivator recognition.