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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Med Chem. Author manuscript; available in PMC Apr 11, 2011.
Published in final edited form as:
PMCID: PMC3073382
NIHMSID: NIHMS251101

Structure of human G protein-coupled receptor kinase 2 in complex with the kinase inhibitor balanol1

Abstract

G protein-coupled receptor kinase 2 (GRK2) is a pharmaceutical target for the treatment of cardiovascular diseases such as congestive heart failure, myocardial infarction, and hypertension. To better understand how nanomolar inhibition and selectivity for GRK2 might be achieved, we have determined the crystal structure of human GRK2 in complex with Gβγ in the presence and absence of the AGC kinase inhibitor balanol. The selectivity of balanol among human GRKs is assessed.

INTRODUCTION

G protein-coupled receptor (GPCR) kinases (GRKsa) phosphorylate activated GPCRs and thereby initiate their uncoupling from heterotrimeric G proteins and their internalization 1, 2. The seven mammalian GRKs (GRK1–7) are grouped into three subfamilies: GRK1 (GRK1 and 7), GRK2 (GRK2 and 3), and GRK4 (GRK4–6).

Although GRK2 is expressed in many tissues, it seems particularly important for embryonic development and heart function, and elevated GRK2 activity is associated with both heart failure and hypertension 3. Thus, it is believed that therapeutic inhibition of GRK2 would improve cardiovascular function. Structural analysis of bovine GRK2 (bGRK2) has led to several crystal structures, including complexes with Gαq and Gβγ heterotrimeric G proteins 46. However, a well-ordered ligand in the active site has not yet been reported, perhaps because in all of these structures the kinase domain adopts an “open”, presumably inactive conformation in which the nucleotide-binding site is not fully formed.

The natural product balanol (Fig. 1a) is a potent, albeit relatively nonselective inhibitor, of AGC kinases 7. X-ray crystal structures of balanol and balanol analogs bound to PKA reveal that the compound binds to a conformation of PKA intermediate to the “open” (inactive) and “closed” (active) states of the kinase 810. Herein we report that GRKs are also potently inhibited by balanol and that the drug exhibits some selectivity among GRK subfamilies. We go on to describe the structure of the human GRK2 (hGRK2)-Gβγ·balanol complex. Comparison with the ligand-free structure reveals that balanol stabilizes the kinase domain of GRK2 in a slightly more closed conformation distinct from that of the PKA·balanol complex.

Figure 1
Balanol and the hGRK2·balanol complex. a) Chemical structure of balanol, with A, B, C, and D rings labeled. b) Balanol bound in the active site of hGRK2. Electron density from a 2.9 Å |Fo|-|Fc| omit map (blue cage) is drawn at the 3σ ...

RESULTS

Inhibition assays

We assessed the ability of balanol to inhibit kinase activity for all seven GRK family members using biotinylated tubulin dimers as a substrate. As shown in Table 1, the IC50 of balanol was ~50 nM for both GRK2 and GRK3, but higher for members of the GRK1 and GRK4 subfamilies. GRK6 was inhibited with the least potency (IC50 ~ 500 nM). The IC50 for PKA under similar conditions was 110 nM (data not shown).

Table 1
Inhibition (IC50, nM) of tubulin phosphorylation by balanol

Structure of the hGRK2-Gβγ complex

hGRK2 and bGRK2 differ by only 11 conservative substitutions, allowing the hGRK2-Gβγ complex to crystallize under the same conditions as bGRK2-Gβγ. The native crystal structure was solved using diffraction data to 2.75 Å spacings (Table S1). hGRK2 and bGRK2 superimpose well, with a root mean squared deviation (rmsd) of 1.1 Å for 612 equivalent Cα positions. The most prominent difference between the two structures is the conformation of the flexible α5–α6 loop region of the regulator of G protein signaling homology domain.

Conformational changes upon binding balanol

The hGRK2-Gβγ·balanol complex was formed by soaking pre-existing crystals or by co-crystallization with balanol (Fig. 1b, Table 2). The resulting crystal structures superimpose with an rmsd of 0.2 Å for 617 Cα positions, indicating that both procedures yield an essentially identical ligand-bound state. The binding of balanol induces conformational changes in the hGRK2 kinase domain (0.6 Å rmsd for 310 Cα atoms between the apo and balanol complexes), including a 4° closure of the large lobe relative to the small lobe around an axis running roughly parallel to the longest dimension of the kinase domain (Fig. 1c). Based on the transition-state-like structure of PKA 11, we estimate that an additional 16° rotation is required to achieve a fully closed state. Subtle conformational changes are also observed within the small lobe (0.5 Å rmsd for 108 Cα atoms). Cα atoms in the β1–β2 (P-loop) and αB–αC loops move up to 1.6 and 1 Å, respectively, away from the active site relative to the apo hGRK2 structure, apparently to help accommodate balanol (Fig. 1c). The large lobe of hGRK2 does not undergo significant conformational changes upon binding balanol (0.3 Å rmsd for 201 Cα atoms).

Comparison with the PKA·balanol complex

Conformational as well as sequence-specific differences between PKA and hGRK2 cause these kinases to bind balanol in distinct ways. Differences in the degree of kinase domain closure cause the pocket accommodating the A ring to be more compressed in GRK2 relative to PKA, whereas the pocket occupied by the benzophenone (C and D) rings appears larger (Fig. 1d). In hGRK2, the A ring is tilted away from the large lobe relative to its position in the PKA·balanol complex, such that its hydroxyl is displaced by 1.4 Å. This is due, in part, to the side chain of hGRK2-Val255, which projects further into the nucleotide binding pocket than the equivalent valine in PKA (Fig. 1d). The position of hGRK2-Val255 is in turn influenced by the more open conformation of the GRK2 kinase domain and a three-residue insertion in the αC-β4 loop (hGRK2 residues 246–255) relative to PKA.

In the hGRK2 structure, balanol binds such that the D ring is positioned up to 1.4 Å closer to the large lobe of the kinase compared to its position in the PKA·balanol complex (Fig. 1d). Consequently, the P-loop of hGRK2, which forms hydrogen bonds and apolar contacts with the benzophenone moiety, is drawn 1.4 Å closer to the large lobe than is the P-loop of PKA. Residues in PKA that contact the benzophenone ring are larger than their equivalents in GRK2, causing the balanol binding site of PKA to appear more compact (Fig. 1d). However, these sequence differences do not appear to greatly diminish the inhibitory potency of balanol.

Balanol itself exhibits significant conformational differences when bound to PKA and hGRK2, demonstrating its ability to adapt to unique active site environments. Rotations are observed in the bonds linking the azepane group (B ring) to the adjacent rings, and in the bond linking the D ring to the rest of the benzophenone moiety.

DISCUSSION

GRKs are atypical members of the AGC kinase family in that their kinase domains require the docking of activated GPCRs to stabilize their most catalytically active state. Indeed, crystal structures of GRK2 4, GRK6 12, and GRK1 13 reveal that in the absence of GPCRs the kinase domains of these proteins exist in a relatively open, presumably inactive conformations compared to the nucleotide bound forms of other AGC kinases 14, 15. Although nucleotides induce a small degree of domain closure in the case of GRK1, the effect is still insufficient to fully coalesce its active site machinery 13. Therefore, one reason balanol exhibits some selectivity among GRKs, but not among the broader AGC kinase subfamily, may be that balanol is required to adapt to the distinct inactive conformations exhibited by these GRK subfamilies, whereas other AGC kinases can more readily adopt conformations similar to that of the PKA·balanol complex.

We docked balanol into structures of GRK1 and GRK6 to identify some potential sources of selectivity (Fig. S1). The binding site for the A ring is more open and perhaps less optimal in GRK1 and GRK6 relative to GRK2. Non-conserved positions in the active site that contact balanol may also influence selectivity. For example, hGRK2-Leu235 in the αC helix directly interacts with the D ring, but is substituted by Gly231 in GRK1 and Met231 in GRK6 (Fig. 1d, S1). Ile197 in the β1 strand directly contacts the A ring but is substituted by leucine in the GRK1 and GRK4 subfamilies.

In PKA, the binding of balanol causes the kinase domain to close by 9°, as opposed to the 4° closure we observe in hGRK2. The resulting balanol-bound conformations of these enzymes are thus distinct. Despite this, balanol is flexible enough to bind with relatively high affinity to either active site. Our results suggest that inhibitors with less flexibility than balanol could be designed to enhance selectivity for GRKs because the inactive states of these enzymes have distinct conformations from each other and from PKA 13. Indeed, drugs that target unique inactive states of other protein kinases have proven remarkably effective 16.

EXPERIMENTAL SECTION

Materials

Balanol was produced by cultivating the fungus strain ST003555 (from Gabon, Africa) on solid rice plates and 10% malt medium at 25 °C for 14 days. After methanol extraction, the proteins were fractionated on MCI® Gel (Mistsubishi Chemical Company; CHP20P, 75–150 µm) and the pooled fractions containing balanol were further purified (>95%) with reverse phase HPLC 17. Biotinylated bovine tubulin dimers were obtained from TEBU-BIO Offenbach, Germany. GRK2–7 proteins for IC50 determination were obtained from Invitrogen, and bovine heart PKA (catalytic subunit) from Sigma. GRK1 was produced by Sanofi-Aventis GmbH.

Inhibition of GRK2-mediated tubulin phosphorylation

GRKs 1–7 with specific activity of 450 pmol/min/mg were first incubated with various concentrations of balanol (100, 20, 4, 0.8, 0.16, 0.032, 0.064, and 0.00128 µM) for 30 min at room temperature in assay buffer (20 mM Tris-HCl pH 7.4, 2 mM EDTA and 2.25% DMSO). The phosphorylation reactions were started by adding MgCl2 (10 mM), ATP (3 µM), [γ-33P]-ATP (0.4 µCi/40 µl) and the GRK·balanol complex to biotinylated bovine tubulin dimers (250 nM) coated on 384 well StreptaWell plates (Roche). The reaction was stopped after 30 min by adding 0.8% BSA, 0.8% Triton X100, 80 mM EDTA, and 400 µM ATP at 4 °C and then incubated for 18 hrs. After a final wash with PBS pH 8.0 the amount of bound 33P-labelled tubulin was quantified by scintillation counting (60 µl Ultima Gold MV scintillation fluid per well) using a Microbeta TriLux microplate scintillation counter (Perkin Elmer).

Purification and crystallization of hGRK2-Gβγ

Geranylgeranylated bovine Gβ1γ2 was expressed in High Five insect cells and purified as previously described 18. hGRK2 was expressed in a 6 liter fermenter using the baculovirus expression system in Sf9 cells and was purified as described previously for bGRK2 18. Crystallization experiments used the second of two peaks that eluted from the Source S cation exchange column, which corresponds to the unphosphorylated form of hGRK2 and 80–85% of the total hGRK2 19.

To produce native crystals, the hGRK2-Gβγ complex was purified by gel filtration chromatography in the presence of 1 mM ATP as described for bGRK2-Gβγ 18. For crystallization, 1 µl of 7.8 mg/ml hGRK2-Gβγ complex and 1 µl of well solution (100 mM MES pH 5.25 or 6.0, 200 mM NaCl, 5% ethylene glycol and 6.3% PEG 3350) were mixed in a drop suspended over 1 mL well solution at 4 °C. Crystals appeared within 10 days and reached maximum dimensions (0.3 × 0.15 × 0.05 mm) in 20 days. Crystals were serially transferred from a harvesting solution containing 100 mM MES pH 6.0, 200 mM NaCl, 10 mM CHAPS, 1 mM ATP, 2 mM MgCl2, 20 mM HEPES pH 8.0 and 1 mM DTT into the same solution omitting ATP and containing in addition 15% ethylene glycol and 10% DMSO in steps of 3.25 and 2.5%, respectively.

For co-crystallization with balanol, the hGRK2-Gβγ complex was purified by gel filtration in the absence of ATP and concentrated to 8.4 mg/ml before adding balanol (50 mM stock in 100% DMSO) to a final concentration of 500 µM. The protein crystallized under similar conditions to the native complex, with the largest crystals obtained in a well solution of 100 mM MES pH 6.0, 200 mM NaCl, 9% PEG 3350. Crystals were harvested in 20 mM HEPES pH 8, 300 mM NaCl, 10 mM CHAPS, 5 mM MgCl2, 12 %PEG3350, 2 mM DTT, 100 mM MES pH 6.0, 250 µM balanol, 0.5 % DMSO, and 25% ethylene glycol.

For soaks with balanol, native crystals were harvested by slow addition of a solution containing 100 mM MES pH 5.6, 300 mM NaCl, 12% PEG 3350, 10 mM CHAPS, 5 mM MgCl2, 20 mM HEPES pH 8.0, 2 mM DTT and 25% ethylene glycol to the hanging drop. The crystals were then serially transferred into harvesting solution that included 250 µM, and then 2.5 mM balanol, giving a final concentration of 5% DMSO. Crystals were then incubated overnight at 4 °C.

Supplementary Material

Fig 1, Table 1

ACKNOWLEDGMENTS

We thank M. Dreyer for the crystal soaking protocol, M. Lohse for the hGRK2 baculovirus, I. Focken for Sf9 expression of hGRK2, and C. Presenti and L. Toti for balanol fermentation and purification. Use of LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (Grant 085P1000817). Use of the Advanced Photon Source was supported by the U. S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-06CH11357.

Footnotes

1Protein coordinates and structure factors for the hGRK2-Gβγ, hGRK2-Gβγ·balanol (soak), and hGRK2-Gβγ·balanol (co-crystal) complexes have been deposited with the Protein Data Bank with PDB codes 3CIK, 3KRW, and 3KRX respectively.

aAbbreviations: AGC kinase, protein kinase A, kinase G and kinase C subfamily; bGRK2, bovine GRK2; DTT, dithiothreitol; G protein, GTP-binding protein; GPCR, G protein-coupled receptor; GRK, GPCR kinase; hGRK2, human GRK2; PKA, protein kinase A; rmsd, root mean squared deviation.

Supporting Information Available: Additional methods, Table S1, and Fig. S1 are provided online. This material is available free of charge via the Internet at http://pubs.acs.org.

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