1. Isolated Regulatory Domains of cGMP-dependent Protein Kinase Iα and Iβ Retain Dimerization and Native cGMP-binding Properties and Undergo Isoform-specific Conformational Changes
- Author
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Kristin A. Higgins, Jackie D. Corbin, Jennifer L. Busch, Robyn Richie-Jannetta, and Sharron H. Francis
- Subjects
Gene isoform ,Sucrose ,Conformational change ,Insecta ,Time Factors ,Protein Conformation ,Molecular Sequence Data ,Allosteric regulation ,Molecular Conformation ,Biochemistry ,Cell Line ,Catalytic Domain ,Centrifugation, Density Gradient ,Cyclic GMP-Dependent Protein Kinases ,Escherichia coli ,Animals ,Humans ,Protein Isoforms ,Amino Acid Sequence ,Protein kinase A ,Cyclic GMP ,Molecular Biology ,Cyclic GMP-Dependent Protein Kinase Type I ,CGMP binding ,Dose-Response Relationship, Drug ,Chemistry ,Wild type ,Cell Biology ,Recombinant Proteins ,Protein Structure, Tertiary ,Kinetics ,Mutagenesis ,Chromatography, Gel ,Biophysics ,Dimerization ,cGMP-dependent protein kinase ,Allosteric Site ,Gene Deletion ,Protein Binding ,Binding domain - 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.
- Published
- 2006