Isolated regulatory domains of cGMP-dependent protein kinase Iα and Iβ retain dimerization and native cGMP-binding properties and undergo isoform-specific conformational changes

Molecular mechanisms that provide for cGMP activation of cGMP-dependent protein kinase (PKG) are unknown. PKGs are dimeric; each monomer contains a regulatory ( R) and catalytic ( C) domain. In this study, isolated recombinant R domains of PKGI alpha(Delta 349-670) and PKGI beta- (Delta 364-685) containing the dimerization and autoinhibitory subdomains and two allosteric cGMP-binding sites were expressed in Sf9 cells. Both R domains were dimers with elongated conformations ( Stokes radii of 44 and 51 A, respectively, and frictional coefficients of 1.6 and 1.8, respectively). Exchange dissociation kinetics and K-D values for cGMP were similar for each holoenzyme and its isolated R domain, indicating that under these conditions the C domain does not appreciably alter cGMP-binding functions of the R domain. As determined by gel filtration chromatography, cGMP binding caused elongation of the PKGI alpha-isolated R domain and contraction of the PKGI alpha-isolated R domain. Cyclic GMP-bound forms of the isoforms have similar physical dimensions that may reflect a common conformation of active isoforms. Elongation of the PKGI beta holoenzyme associated with cGMP binding and PKG activation cannot be explained solely by conformational change in its R domain, but elongation of the PKGI alpha R domain may partially account for the elongation of wild type PKGI alpha associated with cGMP binding. The cGMP-induced conformational changes in the respective R domains are likely to be critical for kinase activation.