This is a default text for your page Jessica Sheehe/Sandbox 1. Click above on edit this page to modify. Be careful with the < and > signs.
You may include any references to papers as in: the use of JSmol in Proteopedia [1] or to the article describing Jmol [2] to the rescue.
I. Introduction
cGMP-dependent protein kinase (PKG) (EC 2.7.11.12) is a member of the AGC kinase family that mediates intracellular signaling through serine and threonine phosphorylation. PKG family members are encoded by two genes: PKGI and PKGII. A splice variant in PKGI leads to expression of PKG-Ia and PKG-Iβ (1). Both PKGI isoforms are found in smooth muscle cells, platelets, cerebellum, and hippocampus, however the PKG-Ia isoform is preferentially located in the heart and lungs as well (2). cGMP, produced from guanylate cyclase through activation by natriuretic peptides or NO, is required for full activation of PKG-Ia (1).
II. Isoforms and Homology
There are two genes that code for PKG—PRKG1 and PRKG2. Alternative splicing of the PRKG1 results in PKG-Ia and PKG-Ib isoforms that differ at their N-terminal region by approximately 65%(1, 3).
III. General Structure
PKG-Ia is a dimer. Each monomer consists of a dimerization domain, an autoinhibitory domain, two cyclic nucleotide binding sites (A site and B site), a switch helical domain, and a catalytic domain (4).
IV. Dimerization Domain
Dimerization of PKG-Ia is mediated through its leucine zipper motif located at residues 9-44 in the N-terminus of the enzyme (5). The zipper also mediates targeting to substrates leading to their phosphorylation (2). Leucine zippers are right-handed helices that interact through their leucine and isoleucine residues forming a left-handed coiled coil. The primary structure is characterized by a heptad repeat of amino acids (abcdefg) where residues a and d are typically hydrophobic and residues e and g are typically hydrophilic. The structure of the PKG-Ia leucine zipper was solved by NMR in 2005 by Schnell and colleagues (PDB 1ZXA). Using COILS and PARCOILS they predicted the parallel structure of the zipper (6). The helical wheel diagram created using DrawCoil1.0 indicates the residues at the dimer interface. In the corresponding sequence, residues in the a and d positions of the heptad repeat are colored green and the e and g positions are colored purple, indicating the predominant residues at the interface are hydrophobic leucines and isoleucines however the structure is also stabilized by hydrophilic residues in the e and g positions that associate through hydrogen bonding and shield the hydrophobic core.
V. Autoinhibitory Site
PKG-Ia contains a autoinhibitory site that spans residues 50-67 and is denoted by the sequence STHIGPRTTRAQGISAEP (7). The site was identified due to its homology to the autoinhibitory site of PKA (8). The residues RAQGISAEP are thought to act as a pseudosubstrate by binding to the catalytic domain in the substrate binding groove to inhibit enzyme activity (9). Binding of the pseudosubstrate is primarily mediated by autophosphorylation (10). Aitken and colleagues identified three autophosphorylation sites on PKG-Ia after incubation with cAMP: Ser50, Thr58, and Ser64 (11). In a study by Busch and colleagues, Ser64 is a conserved residue that is important for autoinhibition and lowering PKG-Ia binding affinity to cGMP, regardless. Mutation to Ala and Thr did not change activity while mutation to Asp or Asn resulted in an almost constitutively active enzyme. These data suggest that autophosphorylation at Ser64 results in stearic hindrance that blocks binding of the pseudosubstrate to the catalytic site and is not due to electrostatic interaction (3). The hydrophobicity of Ile63 was also found to be important for maintaining the inactive state of PKG-Ia. According to Yuasa and colleagues, mutation to Phe, Val, or Leu did not result in a significant decrease in inhibition while mutation to hydrophilic residues such as Gly, Ala, Thr, and Ser abolished inhibitory effects (8).
= VI. Regulatory Domain and Switch Helical Subdomain =
The regulatory domain is composed of two cyclic nucleotide binding sites per monomer of PKG-Ia. The A site includes residues 87-210 binds cAMP or cGMP and is considered the fast binding site. The B site includes residues 211-328 and specificalluy binds cGMP, however is considered the slowing binding site (4, 10). Binding of two cGMP molecules to the regulatory domain leads to activation of the kinase.
The structure of the regulatory domain was determined by X-ray crystallography at 2.5Å resolution (PDB 3SHR). cAMP was found bound to the A site and confirmed the important residues for cyclic nucleotide binding. One oxygen of the phosphate group binds to the backbone amide of Ala169 and to the side chain of Arg176 through electrostatic interactions of 3.2Å and 3.0Å respectively. The second oxygen of the phosphate group of cAMP binds to the backbone amide and side chain of Thr177 with bond distances of 2.7Å and 2.5Å respectively. Additionally, the hydroxyl group of cAMP attached to the sugar hydrogen bonds with the backbone amide of Gly166. These interactions stabilize the binding of cyclic nucleotide to the A site of PKG-Ia (10).
Another interesting feature of PKG-Ia is the switch helical subdomain spanning residues 328-355 (PDB 3SHR). In the switch helical model, the hydrophobic tip of the switch helix in the first protomer binds to a hydrophobic knob of the opposing protomer forming a site of interchain communication that is thought to facilitate crosstalk between the regulatory domains.
== VII. Catalytic Site ==
The exact 3D configuration of the catalytic domain of PKG-Ia is unknown, however it is thought to have a similar shape to PKA. AGC kinases have conserved tertiary structure in their catalytic domains, suggesting that PKG-Ia, as a member of the AGC kinase family, would also conform to a similar 3D structure (12). The catalytic domains of PKG-Ia and PKA have 45% sequence identity according to NCBI’s Protein Blast. In PKA, the N and C lobes sandwich the ATP and substrate for catalysis to occur. Conserved structural features include the glycine rich loop, the catalytic loop, and the activation loop. In PKA, the glycine rich loop contains the sequence motif GxGGxG and forms a pocket for ATP to bind. The catalytic loop motif denoted by the sequence YRDLKxxN is involved in coordinating the phosphotransferase reaction. The activation loop starts with DFG motif and ends with an APE motif (13). The APE sequence is highly conserved among protein kinases (14). Phosphorylation of the activation loop is required for kinase activity. Phosphorylation has been documented on Thr517; mutation of this residue to Ala results in a catalytically dead kinase (15).
=== VIII. Phosphorylation of PKG-Ia ===
According to phosphosite.org, there are eleven previously documented phosphorylations on PKG-Ia: one in the dimerization domain, six in the autoinhibitory site, one in the B site of the regulatory domain, and three in the catalytic domain located on the activation loop. Many of these were discovered by proteomic discovery mass spectrometry only and have not been further characterized. S45 (16), S51 (16, 17), S59 (16, 18, 19), S65 (3, 16, 17, 20), T85 (16, 17), and T517 (15, 21) were discovered by site specific mutagenesis.
==== IX. Evolutionary Conservation ====
The sequence of PKG-Ia is highly conserved among species. Sequence alignment of 12 species (human, bovine, mouse, rat, wild boar, Japanese rice fish, Zebra fish, fruit fly, honey bee, and silk worm) using ClustalW indicated high sequence homology throughout. About 75% of the residues showed invariant or homologous residues. There were 341 invariant residues found in all species, which is about 50% of the entire human PKG-Ia sequence. The region with the most variability was the N-terminus, which interesting has highest region of variability among PKG isoforms.
===== X. PDB Structures =====
