A Crystal Structure of the Cyclic GMP-dependent Protein Kinase Iβ Dimerization/Docking Domain Reveals Molecular Details of Isoform-specific Anchoring*

Abstract

Cyclic GMP-dependent protein kinase (PKG) is a key mediator of the nitric oxide/cGMP signaling pathway and plays a central role in regulating cardiovascular and neuronal functions. The N-terminal ϳ50 amino acids of the kinase are required for homodimerization and association with isoform-specific PKGanchoring proteins (GKAPs), which target the kinase to specific substrates. To understand the molecular details of PKG dimerization and gain insight into its association with GKAPs, we solved a crystal structure of the PKG I␤ dimerization/docking domain. Our structure provides molecular details of this unique leucine/isoleucine zipper, revealing specific hydrophobic and ionic interactions that mediate dimerization and demonstrating the topology of the GKAP interaction surface.As the main effector of the nitric oxide/cGMP signaling cascade, cGMP-dependent protein kinase (PKG) regulates smooth muscle tone, inhibits platelet activation, and modulates neuronal functions (1). In mammalian cells, two different genes encode a soluble type I PKG and a membrane-anchored type II PKG (1). Both enzymes form homodimers through an N-terminal leucine/isoleucine zipper domain. PKG I has two splice variants (␣ and ␤) that differ in the first ϳ100 amino acids, resulting in unique dimerization and autoinhibitory domains. The leucine/isoleucine zipper domain mediates interaction with isotype-specific G-kinase-anchoring proteins (GKAPs), 2 targeting PKG I␣ and I␤ to different subcellular compartments and intracellular substrates (2); therefore, we refer to this region as the dimerization and docking (D/D) domain. The domain organization of PKG is shown in Fig. 1. The N-terminal D/D domain is followed by an inhibitory sequence (IS), tandem cyclic nucleotide binding pockets, and the catalytic domain. The D/D domain contains a distinct primary sequence, with a repeating pattern of leucines and isoleucines every seven residues (Fig. 1). This pattern is referred to as a heptad repeat, and the positions of residues are labeled a-g.Specific binding partners for PKG I␣ include the myosin phosphatase targeting subunit (MYPT1) of myosin light chain phosphatase and the regulator of G-protein signaling-2 (RGS-2) (3, 4). Phosphorylation of MYPT1 by PKG I␣ activates its phosphatase activity, leading to dephosphorylation of myosin light chain, which desensitizes the contractile apparatus response to calcium, resulting in vasorelaxation (3). RGS-2 functions as a GTPase-activating protein for G␣ q subunits of heterotrimeric G-protein complexes, and phosphorylation of RGS-2 by PKG I␣ increases its activity toward G␣ q , uncoupling downstream signaling from G␣ qlinked receptors for vasoconstrictive agents (4). Specific binding partners for PKG I␤ include the inositol triphosphate receptor-associated PKG substrate (IRAG) and the transcriptional regulator TFII-I (5, 6). Phosphorylation of IRAG by PKG I␤ inhibits 1,4,5-inositol triphosphate receptordependent calcium release from the endoplasmic reticulum, contributing to smooth muscle relaxation (7). Phosphor...

IRAG and novel PKG targeting in the cardiovascular system

“…Recent reports described the molecular details of the PKG1␤ dimerization/docking domain upon crystallization of the LZ domain. The structure revealed the topology of this domain to expose specific hydrophobic and ionic interactions mediating dimerization (11). The hydrophilic residues were arranged in a "knobs into holes" fashion.…”

“…C: NH2-terminal 51 amino acids LZ domain of PKG1␤. The orange-boxed amino acids have negative charges (corresponding to the positive-charged amino acids of PKG1␣) that form a negatively charged surface responsible for substrate docking (11). The other marked residues correspond to the functions described in B.…”

“…PKG1␣ and PKG1␤ are derived from the same gene and differ only in the NH2-terminal ϳ100 amino acids regions containing the LZ domains. B: overview of the NH2-terminal 45 amino acids of the PKG1␣ LZ that are postulated to interact as homodimer (11). The amino acids are arranged in a heptad repeat and nearly every a-position has a leucine or isoleucine residue (cyan marked); the majority of d-positions have charged or hydrophilic residues.…”

Crystal Structure of the cGMP-dependent Protein Kinase II Leucine Zipper and Rab11b Protein Complex Reveals Molecular Details of G-kinase-specific Interactions

“…Structures of LZs are reported for both PKG I␣ (PDB 1ZXA) and I␤ (PDB 3NMD) (40,44). As seen in the Fig.…”

“…Superhelical and ␣-helical parameters were analyzed using Twister showing similar values as the previously reported coiled-coil structures, including PKG I␤ LZ (40,41). Both strands exhibit higher temperature factors toward the N and C termini, suggesting increased thermal motion or flexibility in those regions, and show minor distortion based on the ␣-helical parameters by calculated Twister (41).…”