Crystal Structure of G Protein-coupled Receptor Kinase 5 in Complex with a Rationally Designed Inhibitor

Abstract

G protein-coupled receptor kinases (GRKs) regulate cell signaling by initiating the desensitization of active G protein-coupled receptors. The two most widely expressed GRKs (GRK2 and GRK5) play a role in cardiovascular disease and thus represent important targets for the development of novel therapeutic drugs. In the course of a GRK2 structure-based drug design campaign, one inhibitor (CCG215022) exhibited nanomolar IC50 values against both GRK2 and GRK5 and good selectivity against other closely related kinases such as GRK1 and PKA. Treatment of murine cardiomyocytes with CCG215022 resulted in significantly increased contractility at 20-fold lower concentrations than paroxetine, an inhibitor with more modest selectivity for GRK2. A 2.4 Å crystal structure of the GRK5·CCG215022 complex was determined and revealed that the inhibitor binds in the active site similarly to its parent compound GSK180736A. As designed, its 2-pyridylmethyl amide side chain occupies the hydrophobic subsite of the active site where it forms three additional hydrogen bonds, including one with the catalytic lysine. The overall conformation of the GRK5 kinase domain is similar to that of a previously determined structure of GRK6 in what is proposed to be its active state, but the C-terminal region of the enzyme adopts a distinct conformation. The kinetic properties of site-directed mutants in this region are consistent with the hypothesis that this novel C-terminal structure is representative of the membrane-bound conformation of the enzyme.

Keywords: G protein-coupled receptor kinase; crystallography; enzyme inhibitor; membrane targeting; plasma membrane; protein kinase; rational drug design; site-directed mutagenesis; x-ray crystallography.

FIGURE 1.

Rational design of improved GRK inhibitors. Superposition of paroxetine and balanol in complex with GRK2 (PDB entries 3V5W (25) and 3KRW (35), respectively) (A) and of GSK180736A and Takeda103A with GRK2 (PDB entries 4PNK (27) and 3PVW (50), respectively) (B). Paroxetine and GSK180736A are chemically similar molecules that occupy the adenine, ribose, and polyphosphate subsites of the kinase-active site. More potent inhibitors such as balanol and Takeda103A also occupy the hydrophobic subsite, suggesting that extending the GSK180736A scaffold would improve potency of inhibition. Inhibitors are drawn as stick models with carbons colored according to their respective protein structure as follows: oxygens, red; nitrogens, cyan; and fluorines, green. C, chemical structures of GSK180736A and its 2-pyridylmethyl amide derivative CCG215022.

FIGURE 2.

Inhibition of GRKs and PKA by CCG215022. Representative curves from an experiment performed in duplicate in the presence of 5 μm ATP. Symbols are as follows: GRK1 (circles), GRK2 (squares), GRK5 (upward triangles), and PKA (downward triangles). See Table 1 (mean IC₅₀ values in the table are slightly different because they represent aggregate measurements).

FIGURE 3.

Increased adrenergic contractility in myocytes treated with GRK2 inhibitors. A, representative contraction tracings of single adult ventricular cardiomyocytes showing shortening (%) with a basal twitch and after Iso stimulation. Prior to Iso stimulation, a group of cells was pretreated with paroxetine or CCG215022. B, quantitation of maximal single myocyte contraction amplitude under corresponding conditions. *, p < 0.001 versus baseline; #, p < 0.001 versus DMSO Iso.

FIGURE 4.

Crystal structure of GRK5 in complex with CCG215022. A, comparison with the GRK6·sangivamycin complex (PDB entry 3NYN) (13). CCG215022 (spheres with yellow carbons) binds in the active site of the GRK5 kinase domain. As in most other GRK structures, the AST region is disordered (last visible residues denoted by asterisks). The C-terminal region of GRK5 (royal blue) has a dramatically different conformation than observed for GRK6·sangivamycin (brown), despite the fact they are closely related enzymes (see C). Key residues in the C terminus are labeled to emphasize how they contribute to packing in each structure. Side chains shown with beige carbons are from the RH domain of GRK6 (same identity and numbering as in GRK5). GRK6-Pro-547 is analogous to GRK5-Pro-546, which was mutated in this study. B, close-up view of the interactions between the C-terminal region (royal blue) and the RH domain (green). Hydrogen bonds/salt bridges are shown as dashed lines. C, primary structural comparison of GRK4 subfamily members. Secondary structure is indicated above the alignment. Residues observed in any crystal structure are colored black (the structure of GRK4 has not yet been reported), and unresolved residues are gray. Residues involved in the interface between the RH domain and the C-terminal region are highlighted in purple, and the C-terminal CAAX (where A is any aliphatic amino acid and X is any amino acid) box of GRK1 is highlighted in yellow. Although the sequence of GRK4A is the most divergent with respect to residues that form the interface observed in GRK5, it maintains the LXXDL motif, which is likely one of the most critical. GRK1 conserves this motif as MXXDM. Thus, all subfamily members have the potential to form a similar C-terminal structure to that observed in the GRK5·CCG215022 complex.

FIGURE 5.

Ligand interactions in the active sites of GRK5 and GRK2. A, interactions of CCG215022 within the GRK5 active site (cf. Fig. 1A). The inhibitor is drawn with yellow carbons, and black dashed lines depict hydrogen bonds. A 3σ |F_o|−|F_c| omit map for the inhibitor is shown as a magenta wire cage. Comparison of the GRK5·CCG215022 (B) and GRK2·Takeda compound 103A (C) active sites highlights a profound difference in the size of their hydrophobic subsites (black ellipses). Each GRK is rendered as a molecular surface with carbons colored gray, nitrogens colored cyan, and oxygens colored red.

FIGURE 6.

Model of GRK4 subfamily membrane interactions. GRK5 and -6 are proposed to exist in an equilibrium between conformational states that have different membrane binding propensities. In each structure, the C-terminal region is shown in green and the amphipathic αCT helix in red. The rest of the enzyme is shown as spheres. Residues near the N terminus proposed to be involved in binding anionic phospholipids, such as PIP₂ (49), are colored cyan. Based on prior evidence, GRK5 seems to have greater membrane affinity than GRK6 (48), and thus its conformational equilibrium may favor the membrane-bound conformation modeled at top. The soluble form of these enzymes, or at least GRK6, may contain a C-terminal structure similar to that observed in the GRK6·sangivamycin structure (bottom), wherein the hydrophobic surface of the αCT helix packs against the RH domain. In the membrane-bound conformation (top) the αCT helix is modeled as a membrane-binding anchor connected to the rest of the enzyme by a flexible tether (dashed green line). Ca²⁺·CaM binds to the N-terminal PIP₂-binding residues as well as the αCT helix, which dissociates the enzyme from the membrane and allows it to be targeted to the nucleus by a nuclear localization signal within in the large lobe of the kinase domain (15).