Abstract
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 Iα activation by cGMP and cAMP by engineering a monomeric form that lacks N-terminal residues 1-53 (Δ53). 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 Δ53 has a similar shape to the heterodimeric RIα-C complex of PKA. Moreover, we found that the Δ53 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 Iα as assessed by surface plasmon resonance (SPR) spectroscopy. However, we found that the Δ53 variant does not exhibit cooperative activation, and its cyclic nucleotide selectivity is diminished. These findings support a model in which, despite structural similarities, PKG Iα activation is distinct from that of PKA, and its cooperativity is driven by in trans interactions between protomers.
Highlights
The type I cGMP-dependent protein kinases (PKG I) serve essential physiological functions, including smooth muscle relaxation, cardiac remodeling, and platelet aggregation
Using the ⌬53 construct, we address the following: 1) whether PKG I␣ is inhibited in cis or in trans, 2) how cyclic nucleotide binding and selectivity of the A-site is linked to activation, and 3) a putative mechanism by which cGMP-mediated cooperativity is derived in PKG
The minor phosphorylation site observed for ⌬53 was suspected to reside in the autoinhibitory domain [35, 36]
Summary
The design of a monomeric form of PKG I␣ was accomplished using a bioinformatics approach by comparing with a previously solved structure of the homolog, PKA, in a heterodimeric complex with its regulatory domain (Fig. 1, A and B). Tandem MS analyses of trypsin-digested samples were used to confirm the primary phosphorylation site at Thr517 (full-length enzyme numbering scheme) located in the activation loop of the catalytic domain (Table 1). To characterize the low-resolution solution structure of the autoinhibited complex, we utilized sizeexclusion chromatography (SEC) coupled to SAXS wherein during analysis a single species associated with ⌬53 was observed (Table 2). Predicted X-ray scattering from the crystal structures of the PKA RI␣–C, PKA RII–C, and PfPKG from Plasmodium falciparum (a monomeric species containing four cGMP-binding sites and a catalytic domain) was conducted using both CRYSOL and FoXS (Table 2f) [38, 39]. The best fit of the experimental SAXS data to the predicted scattering was obtained from RI␣–C using CRYSOL with constant subtraction (2 ϭ 0.82; Fig. 3D and Table 2). Using SUPCOMB, the resulting averaged and filtered DAMMIF/DAMMIN ab initio three-dimensional envelope
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