title: Isolated regulatory domains of cGMP-dependent protein kinase Ialpha and Ibeta retain dimerization and native cGMP-binding properties and undergo isoform-specific conformational changes.
abstract: 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 PKGIalpha-(Delta349-670) and PKGIbeta-(Delta364-685) 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 PKGIalpha-isolated R domain and contraction of the PKGIbeta-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 PKGIbeta holoenzyme associated with cGMP binding and PKG activation cannot be explained solely by conformational change in its R domain, but elongation of the PKGIalpha R domain may partially account for the elongation of wild type PKGIalpha associated with cGMP binding. The cGMP-induced conformational changes in the respective R domains are likely to be critical for kinase activation.
authors.affiliation: Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN 37232-0615, USA.
authors.forename: Robyn
authors.initials: R
authors.lastname: Richie-Jannetta
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Busch
authors.forename: Kristin A
authors.initials: KA
authors.lastname: Higgins
authors.forename: Jackie D
authors.initials: JD
authors.lastname: Corbin
authors.forename: Sharron H
authors.initials: SH
authors.lastname: Francis
mesh_terms: D000495:Allosteric Site; D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D006152:Cyclic GMP / Q000737:chemistry / Q000378:metabolism; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases / Q000737:chemistry* / Q000378:metabolism; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D017353:Gene Deletion; D006801:Humans; D007313:Insecta; D007700:Kinetics; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011485:Protein Binding; D011487:Protein Conformation; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D013395:Sucrose / Q000494:pharmacology; D013997:Time Factors
chemical_list: D020033:Protein Isoforms; D011994:Recombinant Proteins; D013395:Sucrose; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases; C569718:PRKG1 protein, human; D006152:Cyclic GMP