Activation by cyclic GMP binding causes an apparent conformational change in cGMP-dependent protein kinase

Cyclic nucleotide binding activates cyclic nucleotide-dependent protein kinases, but the molecular mechanism is unknown. In the present studies, cGMP binding to type I alpha or type I beta cGMP-dependent protein kinase (PKG) caused (i) a large electronegative charge shift of each enzyme on ion exchange chromatography, (ii) an increase in the Stokes radius (>3 Angstrom) of each enzyme, and (iii) a decreased mobility of type I beta PKG on native gel electrophoresis, These physical changes were not detected in the monomeric form of type I beta PKG upon activation by cGMP, However, the results of partial proteolysis of type I alpha PKG revealed some degree of cGMP-induced conformational change within the PKG-monomer, since cGMP binding protects the PKG-monomer against chymotryptic cleavage, The altered sensitivity to proteolysis occurs at Met-200, which is located between the B and C alpha-helices in the high affinity site (site A), and implies that the cGMP-induced structural perturbations in this region may participate in activation of dimeric PKG, The cGMP-induced conformational effects observed using the physical separation methods are likely to reflect altered interactions within the dimeric PKG that are caused by structural alterations within the subunits.