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. Copyright © 1995 The Protein Society