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Exploring the interaction between the protein kinase A catalytic subunit and caveolin-1 scaffolding domain with shotgun scanning, oligomer complementation, NMR, and docking

¹ Department of Chemistry and ² Department of Molecular Biology and Biochemistry, University of California, Irvine, California 92697, USA

(RECEIVED October 13, 2005; FINAL REVISION December 3, 2005; ACCEPTED December 9, 2005)

The techniques of phage-displayed homolog shotgun scanning, oligomer complementation, NMR secondary structure analysis, and computational docking provide a complementary suite of tools for dissecting protein–protein interactions. Focusing these tools on the interaction between the catalytic sub-unit of protein kinase A (PKA_cat) and caveolin-1 scaffolding domain (CSD) reveals the first structural model for the interaction. Homolog shotgun scanning varied each CSD residue as either a wild-type or a homologous amino acid. Wild-type to homolog ratios from 116 different homologous CSD variants identified side-chain functional groups responsible for precise contacts with PKA_cat. Structural analysis by NMR assigned an -helical conformation to the central residues 84– 97 of CSD. The extensive mutagenesis data and NMR secondary structure information provided constraints for developing a model for the PKA_cat–CSD interaction. Addition of synthetic CSD to phage-displayed CSD resulted in oligomer complementation, or enhanced binding to PKA_cat. Together with previous experiments examining the interaction between CSD and endothelial nitric oxide synthase (eNOS), the results suggest a general oligomerization-dependent enhancement of binding between signal transducing enzymes and caveolin-1.

Keywords: caveolin; phage display; mutagenesis; protein kinase A; molecular recognition; stability and mutagenesis; specificity; structure/function studies; membrane-associated proteins; NMR spectroscopy; docking proteins; computational modeling

Abbreviations: CSD, caveolin-1 scaffolding domain • DQF-COSY, double quantum filtered correlation spectroscopy • eNOS, endothelial nitric oxide synthase • NOESY, nuclear Overhauser effect spectroscopy • NOE, nuclear Overhauser effect • PKA_cat, protein kinase A catalytic subunit • PKI, protein kinase inhibitor • TOCSY, total correlations spectroscopy

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051911706.

Reprint requests to: Gregory A. Weiss, Department of Chemistry, University of California, Irvine, CA 92697, USA; e-mail: gweiss{at}uci.edu; fax: (949) 824-9920.

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