The objectives of this proposal are to probe the structure and function of the type I regulatory (R) subunit of cAMP-dependent protein kinase. Ultimately our goals are to understand the molecular basis for cAMP binding, for interaction with the catalytic (C) subunit, and for cAMP- mediated dissociation of the holoenzyme. The R-subunit exists in two conformational states. In the absence of cAMP, it forms an inactive tetrameric holoenzyme that, in the case of the type I holoenzyme, requires the high affinity binding of MgATP. Cyclic AMP dissociates the complex into an R2(cAMP)4 dimer and two active C-subunits. The R- subunit has a well-defined domain structure with a dimerization domain at the N-terminus followed by an autoinhibitor site that occupies the peptide binding site in the holoenzyme. Two tandem cAMP-binding domains lie at the C-terminus. The R structure will be probed in several ways. CAMP-binding sites, R-C interaction sites, and MgATP-interaction sites will be probed by site-directed mutagenesis. Selective cysteines will be engineered into the RI-subunit for the attachment of fluorescent probes in order to map R-C interaction sites and to probe cAMP and C- induced conformational changes. Isolated domain will also be prepared. cAMP-binding domain B will be characterized not only for its functional properties, but also for its capacity to reassemble with the remaining part of the protein to form an intact, functional R-subunit. Conformational changes will be followed using NMR techniques after first labeling with 15N in E. coli. Delta-I-91 deletion mutants containing point mutations in each cAMP-binding site will also be 15N- labeled. In addition to the cAMP-binding domains, the stable dimerization domain will be characterized. Preliminary results suggest that this is a stable helix bundle linked by two interchain disulfide bonds and heterodimers occur in vivo. The dimerization domain will be expressed and/or isolated by CNBr cleavage. Homo and heterodomains will be probed using NMR and crystallography. Heterodimers and homodimers between RI-alpha and RI-beta will be characterized. A major goal during this next granting period will be to obtain a high-resolution crystal structure of the R-subunit and various mutant forms of the R-subunit. Crystallization of the holoenzyme complex will also be a long-term goal.
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