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A simple electrostatic switch important in the activation of type I protein kinase A by cyclic AMP

¹ Department of Chemistry and Biochemistry, and ² Howard Hughes Medical Institute, University of California, San Diego (UCSD), San Diego, California 92037, USA

(RECEIVED July 28, 2005; FINAL REVISION October 19, 2005; ACCEPTED October 19, 2005)

Cyclic AMP activates protein kinase A by binding to an inhibitory regulatory (R) subunit and releasing inhibition of the catalytic (C) subunit. Even though crystal structures of regulatory and catalytic subunits have been solved, the precise molecular mechanism by which cyclic AMP activates the kinase remains unknown. The dynamic properties of the cAMP binding domain in the absence of cAMP or C-subunit are also unknown. Here we report molecular-dynamics simulations and mutational studies of the RI R-subunit that identify the C-helix as a highly dynamic switch which relays cAMP binding to the helical C-subunit binding regions. Furthermore, we identify an important salt bridge which links cAMP binding directly to the C-helix that is necessary for normal activation. Additional mutations show that a hydrophobic "hinge" region is not as critical for the cross-talk in PKA as it is in the homologous EPAC protein, illustrating how cAMP can control diverse functions using the evolutionarily conserved cAMP-binding domains.

Keywords: conformational changes; structure/function studies; molecular mechanics/dynamics; site-directed mutagenesis; ligand binding

Abbreviations: PKA, protein kinase A or cyclic AMP-dependent protein kinase • cAMP, cyclic adenosine monosphosphate • cGMP, cyclic guanosine monophosphate • ATP, adenosine triphosphate • C-subunit, catalytic subunit • R-subunit, regulatory subunit • PBC, phosphate binding cassette • CD, circular dichroism • K_a, activation constant • RMSD, root-mean-square distance • EPAC, exchange protein directly activated cAMP

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051723606.

Reprint requests to: Susan S. Taylor, Department of Chemistry and Biochemistry, University of California, San Diego (UCSD), MC 0654, San Diego, CA 92037, USA; e-mail: staylor{at}ucsd.edu; fax: (858) 534-8193.

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