DISSECTING CAMP BINDING DOMAIN-A IN THE R-I-ALPHA SUBUNIT OF CAMP-DEPENDENT PROTEIN-KINASE - DISTINCT SUBSITES FOR RECOGNITION OF CAMP AND THE CATALYTIC SUBUNIT

The two gene-duplicated cAMP binding domains in the regulatory subunit s of cAMP dependent protein kinase are each comprised of an A helix, a n eight-stranded beta-barrel, and a B and C helix (1). The A domain is required for high affinity binding to C, while the B domain regulates access to the A domain. Using a combination of a yeast two-hybrid scr een coupled with deletion analysis, cAMP binding domain A of R-1 was d issected into two structurally and functionally distinct subsites, one that binds cAMP and another that binds the C subunit, The minimum sta ble subdomain required for binding to C in the 1-3 micromolar range is composed of residues 94-169, while residues 236-244, mapped to the C helix of cAMP binding domain A, were defined as a second surface neces sary for high affinity (5-10 nanomolar) binding to C. This portion of the C helix, due to its position directly between the two subsites, se rves as a molecular switch for either a cAMP-bound conformation or a C -bound conformation and can thus modulate interactions of cAMP binding domain A with cAMP, with C, and with cAMP binding domain B.