Effects of cAMP-binding site mutations on intradomain cross-communication in the regulatory subunit of cAMP-dependent protein kinase I (original) (raw)

1990, Journal of Biological Chemistry

Each protomer of the regulatory subunit dimer of CAMP-dependent protein kinase contains two tandem and homologous CAMP-binding domains, A and B, and cooperative CAMP binding to these two sites promotes holoenzyme dissociation. Several amino acid residues in the type I regulatory subunit, predicted to lie in close proximity to each bound cyclic nucleotide based on affinity labeling and model building, were replaced using recombinant techniques. The mutations included replacement of 1) Glu-200, predicted to hydrogen bond to the 2'-OH of CAMP bound to site A, with Asp, 2) Tyr-371, the site of affinity labeling with 8-N3-CAMP in site B, with Trp, and 3) Phe-247, the position in site A that is homologous to Tyr-371 in site B, with Tyr. Each mutation caused an approximate a-fold increase in both the K,(cAMP) and &(cAMP); however, the offrates for CAMP and the characteristic pattern of affinity labeling with S-N3-CAMP differed markedly for each mutant protein. Furthermore, these mutations affect the CAMP binding properties not only of the site containing the mutation, but of the adjacent nonmutated site as well, thus confirming that extensive crosscommunication occurs between the two CAMP-binding domains.