Characterization of the isolated cAMP‐binding B domain of cAMP‐dependent protein kinase

Abstract

A 14.4-kDa cAMP-binding fragment was generated during bacterial expression and purification of recombinant bovine cAMP-dependent protein kinase type Iα regulatory subunit (RIα). The full-length RIα from which the fragment was derived contained a point mutation allowing its B domain to bind both cAMP and cGMP with high affinity while leaving its A domain highly cAMP selective. The NH₂ terminus of the fragment was Ser-252, indicating that it encompassed the entire predicted B domain. Although the [³H]cAMP and [³H]cGMP exchange rates of the isolated B domain were increased relative to the B domain in intact RIα, the [³H]cAMP exchange rate was comparable to that of the B domain of full-length RIα containing an unoccupied A domain. A plasmid encoding only the isolated B domain was overexpressed in Escherichia coli, and a monomeric form of the B domain was purified that had identical properties to the proteolytically generated fragment, indicating that all of the elements for the high-affinity cAMP-binding B domain are contained within the 128 amino acid carboxyl terminus of the R subunit. Prolonged induction of the B domain in E. coli or storage of the purified protein resulted in the formation of a dimer that could be reverted to the monomer by incubation in 2-mercaptoethanol. Dimerization caused an approximate fivefold increase in the rate of cyclic nucleotide exchange relative to the monomer. The results show that an isolated cAMP-binding domain can function independently of any other domain structures of the R subunit.