The catalytic domain of the cGMP-dependent protein kinase Iα modulates the cGMP-binding characteristics of its regulatory domain

AbstractThe cGMP-dependent protein kinase Iα (PKG Iα) possesses two functional moieties, the regulatory and catalytic domains, which reside on a single polypeptide chain. Here we report on the influence of the catalytic domain on the binding of cGMP to the regulatory domain. A deletion mutant, Δ352–670 of PKG Iα, lacking the catalytic domain, was constructed and expressed in E. coli. The purified 38 kDa mutant protein showed strong reactivity toward tryptic proteolysis at residue Arg77. Thus, a double deletion fragment Δ1–77/352–670 PKG Iα, lacking the N-terminus, was also purified. Both proteins had functional cGMP binding, but differed kinetically from the wild-type protein. First the affinity constants for cGMP were modulated, second the constructs showed no signs of cooperative cGMP binding and third dimerization of the Δ352–670 mutant was abolished. Our results provide evidence that the catalytic domain forms an intimate interaction with the regulatory domain and modulates the kinetics of cGMP binding