Search

As one of the main receptors of a second messenger, cGMP, cGMP-dependent protein kinase (PKG) isoforms I and II regulate distinct physiological processes. The design of isoform-specific activators is thus of great biomedical importance and requires detailed structural information about PKG isoforms bound with activators, including accurate positions of hydrogen atoms and a description of the hydrogen bonding and water architecture. Here, we determined a 2.2 Å room-temperature joint X-ray/neutron (XN) structure of the human PKG II carboxyl cyclic nucleotide binding (CNB-B) domain bound with a potent PKG II activator, 8-pCPT-cGMP. The XN structure directly visualizes intermolecular interactions and reveals changes in hydrogen bonding patterns upon comparison to the X-ray structure determined at cryo-temperatures. Comparative analysis of the backbone hydrogen/deuterium exchange patterns in PKG II:8-pCPT-cGMP and previously reported PKG Iβ:cGMP XN structures suggests that the ability of these agonists to activate PKG is related to how effectively they quench dynamics of the cyclic nucleotide binding pocket and the surrounding regions.

Cyclic GMP analogs, 8-Br, 8-pCPT, and PET-cGMP, have been widely used for characterizing cellular functions of cGMP-dependent protein kinase (PKG) I and II isotypes. However, interpreting results obtained using these analogs has been difficult due to their low isotype specificity. Additionally, each isotype has two binding sites with different cGMP affinities and analog selectivities, making understanding the molecular basis for isotype specificity of these compounds even more challenging. To determine isotype specificity of cGMP analogs and their structural basis, we generated the full-length regulatory domains of PKG I and II isotypes with each binding site disabled, determined their affinities for these analogs, and obtained cocrystal structures of both isotypes bound with cGMP analogs. Our affinity and activation measurements show that PET-cGMP is most selective for PKG I, whereas 8-pCPT-cGMP is most selective for PKG II. Our structures of cyclic nucleotide binding (CNB) domains reveal that the B site of PKG I is more open and forms a unique π/π interaction through Arg285 at β4 with the PET moiety, whereas the A site of PKG II has a larger β5/β6 pocket that can better accommodate the bulky 8-pCPT moiety. Our structural and functional results explain the selectivity of these analogs for each PKG isotype and provide a starting point for the rational design of isotype selective activators.

The complete amino acid sequence of the cyclic GMP stimulated cyclic nucleotide phosphodiesterase (cGS-PDE) of bovine heart has been determined by analysis of five digests of the protein; placement of the C-terminal 330 residues has been confirmed by interpretation of the corresponding partial cDNA clone. The holoenzyme is a homodimer of two identical N alpha-acetylated polypeptide chains of 921 residues, each with a calculated molecular weight of 103,244. The C-terminal region, residues 613-871, of the cGS-PDE comprises a catalytic domain that is conserved in all phosphodiesterase sequences except those of PDE 1 from Saccharomyces cerevisiae and a secreted PDE from Dictyostelium. A second conserved region, residues 209-567, is homologous to corresponding regions of the alpha and alpha' subunits of the photoreceptor phosphodiesterases. This conserved domain specifically binds cGMP and is involved in the allosteric regulation of the cGS-PDE. This regulatory domain contains two tandem, internal repeats, suggesting that it evolved from an ancestral gene duplication. Common cyclic nucleotide binding properties and a distant structural relationship provide evidence that the catalytic and regulatory domains within the cGS- and photoreceptor PDEs are also related by an ancient internal gene duplication.

A 'H NMR study on the Escherichia coli aden- osine cyclic 3',5'-phosphate receptor protein (CRP) alone and in its complexes with adenosine cyclic 3',5'-phosphate (CAMP), guanosine cyclic 3',5'-phosphate (cGMP), and a number of analogues is presented. Five sets of imidazole proton reso- nances are seen, corresponding to the five histidines per subunit found by amino acid analysis (Anderson, W. B., Schneider, A. B., Emmer, M., Perlman, R. L., & Pastan, I. (1971) J. Biol. Chem. 246, 592949371, Four of the histidine residues, A, B, C, and D, are characterized by imidazole proton resonances with unusually narrow line widths (3-5 Hz) for a protein of molecular weight 45 000 and pKs in the range 6-7, suggesting that they lie in mobile regions of the protein. In contrast, the fifth histidine residue, E, has a broad C(2) proton resonance (- 15 Hz) and a very low pK (55), indicating that it is buried in the deprotonated state in a rigid portion of the protein. The addition of cyclic nucleotides to CRP does not lead to any drastic changes in the protein spectrum. The most prominent changes are associated with the addition of cAMP and are characterized by an overall broadening of all the proton res- onances of the protein and marked changes at the high-field end of the aromatic region. Following the addition of cAMP and cGMP, a slow conformational change in the structure of CRP can be detected. This conformational transition occurs subsequent to the binding of a cyclic nucleotide molecule to one of the binding sites and is only complete when both cyclic nucleotide binding sites are almost completely saturated. For the cGMPCRP complex, the upper limit for the intercon- version rate between the two conformations is 18 s-I, and for the cAMP.CRP complex, the interconversion rate lies in the range 60-120 s-'. The conformations about the glycosidic bond of cyclic nucleotides bound to CRP were investigated by the measurement of transferred nuclear Overhauser en- hancements, which showed that cAMP and its analogues are bound in the syn conformation with values for the glycosidic bond torsion angle x (0(4')-C( l')-N(9)-C(4)) in the range 45-55' and that cGMP and its analogue inosine cyclic 3',5'-phosphate (cIMP) are bound in the anti conformation with values of x of 225' and 240°, respectively. This contrasts to the situation in free cyclic nucleotides which exist in a syn/anti equilibrium mixture, cAMP being predominantly in the anti conformation and cGMP predominantly in the syn conformation. The implications and possible mechanism of this conformational selection by CRP are discussed. the glycosidic bond of a number of bound cyclic nucleotides including cAMP and cGMP. We show that whereas cAMP and its analogous are bound in the syn conformation, cGMP and its analogues are bound in the anti conformation. This contrasts with their conformations in free solution where cyclic