A Generalized Allosteric Mechanism for cis-Regulated Cyclic Nucleotide Binding Domains

Cyclic nucleotides (cAMP and cGMP) regulate multiple intracellular processes and are thus of a great general interest for molecular and structural biologists. To study the allosteric mechanism of different cyclic nucleotide binding (CNB) domains, we compared cAMP-bound and cAMP-free structures (PKA, Epac, and two ionic channels) using a new bioinformatics method: local spatial pattern alignment. Our analysis highlights four major conserved structural motifs: 1) the phosphate binding cassette (PBC), which binds the cAMP ribose-phosphate, 2) the “hinge,” a flexible helix, which contacts the PBC, 3) the β2,3 loop, which provides precise positioning of an invariant arginine from the PBC, and 4) a conserved structural element consisting of an N-terminal helix, an eight residue loop and the A-helix (N3A-motif). The PBC and the hinge were included in the previously reported allosteric model, whereas the definition of the β2,3 loop and the N3A-motif as conserved elements is novel. The N3A-motif is found in all cis-regulated CNB domains, and we present a model for an allosteric mechanism in these domains. Catabolite gene activator protein (CAP) represents a trans-regulated CNB domain family: it does not contain the N3A-motif, and its long range allosteric interactions are substantially different from the cis-regulated CNB domains

Expression of a chimeric, cGMP-sensitive regulatory subunit of the cAMP-depedent protein kinase type Iα

AbstractTo study the fluctuations of cGMP in living cells through changes of energy transfer of dissociable fluorescence labeled subunits, we constructed a cGMP-sensitive probe by combining the N-terminus of the type I regulatory subunit of cAMP-dependent protein kinase (PKA) with the cGMP binding sites of cGMP-dependent protein kinase Iα (PKG). This chimeric regulatory subunit retained PKA-like dimerization and PKG-compatible cGMP binding constants (Kd = 53 nM) for both binding sites. High affinity interaction with the PKA catalytic subunit was verified by Surface Plasmon Resonance (Kd = 3.15 nM). Additionally, the chimera inhibits the formation of wild-type holoenzyme with an apparent Ki of 1.05 nM. Furthermore, cGMP dissociated the mutant holoenzyme with an apparent activation constant of 146 nM. Thus, our construct provides all the requirements needed to investigate changes in intracellular cGMP concentrations

Differential patterning of cGMP in vascular smooth muscle cells revealed by single GFP-linked biosensors

Here, we report the design of unprecedented, non-FRET based cGMP-biosensors, named FlincGs, to assess the dynamics of nitric oxide (NO) and atrial natriuretic peptide (ANP) induced synthesis of intracellular cGMP, [cGMP]i. Regulatory fragments of PKG I α, PKG I β, and an N-terminal deletion mutant of PKG I α were fused to circular permutated EGFP to generate α-, β-, and δ-FlincG, with high dynamic ranges and apparent KD,cGMP values of 35 nM, 1.1 μM, and 170 nM, respectively. All indicators displayed significant selectivity for cGMP over cAMP, and 1.5- to 2.1-fold increases in fluorescence intensity at 510 nm when excited at 480 nm. Surprisingly, FlincGs displayed an additional excitation peak at 410 nm. δ-FlincG permitted ratiometric (480/410 nm) measurements, with a cGMP-specific 3.5-fold ratio change. In addition, δ-FlincG presented cGMP association and dissociation kinetics sufficiently fast to monitor rapid changes of [cGMP]i in intact cells. In unpassaged, adenoviral transfected vascular smooth muscle (VSM) cells, δ-FlincG had an EC50,cGMP of 150 nM, and revealed transient global cGMP elevations to sustained physiological NO (EC50,DEA/NO = 4 nM), and the decay phase depended on the activity of PDE-5. In contrast, ANP elicited sustained submembrane elevations in [cGMP]i, which were converted to global cGMP elevations by inhibition of PDE-5 by sildenafil. These results indicate that FlincG is an innovative tool to elucidate the dynamics of a central biological signal, cGMP, and that NO and natriuretic peptides induce distinct cGMP patterning under the regulation of PDE-5, and therefore likely differentially engage cGMP targets

An N-terminally truncated form of cyclic GMP–dependent protein kinase Iα (PKG Iα) is monomeric and autoinhibited and provides a model for activation

The type I cGMP-dependent protein kinases (PKG I) serve essential physiological functions, including smooth muscle relaxation, cardiac remodeling, and platelet aggregation. These enzymes form homodimers through their N-terminal dimerization domains, a feature implicated in regulating their cooperative activation. Previous investigations into the activation mechanisms of PKG I isoforms have been largely influenced by structures of the cAMP-dependent protein kinase (PKA). Here, we examined PKG I activation by cGMP and cAMP by engineering a monomeric form that lacks N-terminal residues 1-53 (53). We found that the construct exists as a monomer as assessed by whole-protein MS, size-exclusion chromatography, and small-angle X-ray scattering (SAXS). Reconstruction of the SAXS 3D envelope indicates that 53 has a similar shape to the heterodimeric RI-C complex of PKA. Moreover, we found that the 53 construct is autoinhibited in its cGMP-free state and can bind to and be activated by cGMP in a manner similar to full-length PKG I as assessed by surface plasmon resonance (SPR) spectroscopy. However, we found that the 53 variant does not exhibit cooperative activation, and its cyclic nucleotide selectivity is diminished. These findings support a model in which, despite structural similarities, PKG I activation is distinct from that of PKA, and its cooperativity is driven by in trans interactions between protomers.United States Department of Energy (DOE), Office of Science, Office of Basic Energy Sciences [DE-AC02-76SF00515]; DOE Office of Biological and Environmental Research; National Institute of General Medical Sciences at the National Institutes of Health (NIH) [P41GM103393]; Centers of Biomedical Research Excellence (COBRE) from the NIGMS, National Institutes of Health [P30-GM118228]12 month embargo; published online: 30 March 2018This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]