High-resolution structure of RGS17 suggests a role for Ca2+ in promoting the GTPase-activating protein activity by RZ subfamily members

Abstract

Regulator of G protein signaling (RGS) proteins are negative regulators of G protein-coupled receptor (GPCR) signaling through their ability to act as GTPase-activating proteins (GAPs) for activated Gα subunits. Members of the RZ subfamily of RGS proteins bind to activated Gα_o, Gα_z, and Gα_i1-3 proteins in the nervous system and thereby inhibit downstream pathways, including those involved in Ca²⁺-dependent signaling. In contrast to other RGS proteins, little is known about RZ subfamily structure and regulation. Herein, we present the 1.5-Å crystal structure of RGS17, the most complete and highest-resolution structure of an RZ subfamily member to date. RGS17 cocrystallized with Ca²⁺ bound to conserved positions on the predicted Gα-binding surface of the protein. Using NMR chemical shift perturbations, we confirmed that Ca²⁺ binds in solution to the same site. Furthermore, RGS17 had greater than 55-fold higher affinity for Ca²⁺ than for Mg²⁺ Finally, we found that Ca²⁺ promotes interactions between RGS17 and activated Gα and decreases the K_m for GTP hydrolysis, potentially by altering the binding mechanism between these proteins. Taken together, these findings suggest that Ca²⁺ positively regulates RGS17, which may represent a general mechanism by which increased Ca²⁺ concentration promotes the GAP activity of the RZ subfamily, leading to RZ-mediated inhibition of Ca²⁺ signaling.

Keywords: G protein-coupled receptor (GPCR); GTPase activating protein (GAP); RGS17; RZ subfamily; calcium; cell signaling; crystal structure; heterotrimeric G protein; isothermal titration calorimetry (ITC); nuclear magnetic resonance (NMR); protein crystallization; regulator of G protein signaling (RGS).

Figure 1.

Crystal structure of RGS17 in complex with Ca²⁺. RGS17 crystallized as a dimer but is monomeric in solution as determined by size-exclusion chromatography and NMR. Chain X is shown color-ramped from blue at the N terminus to red at the C terminus. Chain A is shown in gray, and the r.m.s.d. between chains is 0.6 Å. Strong electron density was observed for four Ca²⁺ ions (black spheres). The 10 σ |F_o| − |F_c| omit maps for the Ca²⁺ ions are shown as blue cages, and electron density for these ions persists beyond 20 σ.

Figure 2.

Superposition of RGS17 and 1ZV4 (21). The bundle subdomains of RGS17–Ca²⁺ (cyan; Chain X) and 1ZV4 (fuchsia) (21) have an r.m.s.d. of 0.52 Å for the Cα atoms. The RGS17–Ca²⁺ terminal subdomain is rotated ∼17° from the bundle subdomain with respect to its orientation in the 1ZV4 structure. The N and C termini of the proteins are labeled N and C. The 1ZV4 structure includes residues 66–143 and 146–204.

Figure 3.

The crystallographic RGS17 dimer coordinates four Ca²⁺ ions. RGS17 chains are colored as in Fig. 1. Ca²⁺ ions are shown as black spheres, waters are shown as red spheres, and the distance between the Ca²⁺ ion and the coordinating atoms are shown in dashed yellow lines. All coordination distances are between 2.3 and 2.6 Å. A, the backbone carbonyl oxygen of Tyr-106 and side chain of Glu-109 in the α3–α4 loop coordinate one Ca²⁺ ion in Chain X. This is in close proximity to Ser-150, the GAP residue in the RZ subfamily. B, as observed in A, RGS17 Chain A also coordinates Ca²⁺ via Tyr-106 and Glu-109. A second Ca²⁺ is bound by the backbone carbonyl of Ile-143 and side chain of Glu-148 in the α5–α6 loop. C, the carbonyl oxygen of Gln-124, located in the α4–α5 loop, coordinates a Ca²⁺ ion in Chain X.

Figure 4.

RGS17 binds Ca²⁺ in solution. A, ¹H-¹⁵N 2D HSQC spectra of RGS17 alone (black) or upon addition of 15 mm CaCl₂ (red). B, structure of RGS17 where residues that display chemical shift perturbations greater than 0.15 ppm are shown as ball-and-stick in red. Ca²⁺ is shown as a black sphere. C, graph of CSPs for all the residues that could be assigned in the ¹H-¹⁵N 2D HSQC spectra for RGS17.

Figure 5.

RGS17 binds Mg²⁺ in solution. A, ¹H-¹⁵N 2D HSQC spectra of RGS17 alone (black) or upon addition of 15 mm MgCl₂ (red). B, structure of RGS17 where residues with CSPs greater than 0.15 ppm are shown in ball-and-stick in red. In contrast to spectra obtained in the presence of CaCl₂, MgCl₂ induces CSPs in two regions of RGS17. The locations of the Mg²⁺ ions (black spheres) are modeled based on the location of Ca²⁺ atoms observed in the RGS17–Ca²⁺ crystal structure. C, graph of CSPs for all the residues that could be assigned in the ¹H-¹⁵N 2D HSQC spectra for RGS17.

Figure 6.

RGS17 binds Ca²⁺ with higher affinity than Mg²⁺. The K_D,avg for Ca²⁺ and Mg²⁺ binding to RGS17 was determined for each amino acid that displayed a significant CSP upon the addition of divalent cation. Two cation-binding sites were identified on RGS17, one formed by Tyr-106 and Glu-109 and a secondary site formed by Ile-143 and Glu-148. Residues adjacent to these binding sites that displayed CSPs >2 S.D. greater than the average CSP were used to calculate the K_D,avg for each site by fitting the CSP as a function of ion concentration to a one-site binding model. A, CSP as a function of increasing Ca²⁺ concentration. The K_D,avg for residues Ser-107, Glu-108, Glu-109, and Asn-110 is 132 ± 35 μm, and the K_D,avg for residues Ser-145 and Val-149 is 91 ± 6 μm. B, CSP as a function of increasing Mg²⁺ concentration. The K_D,avg for residues Ser-107, Glu-108, Glu-109, and Asn-110 is 34 ± 23 mm, and the K_D,avg for residues Ser-145 and Val-149 is 20 ± 4 mm.

Figure 7.

RGS4 binds Ca²⁺ weakly but does not bind Mg²⁺. RGS4 shares 40% sequence identity with RGS17, including the residues that bind cations in RGS17. In RGS4, these sites contain Tyr-84 and Glu-86 (RGS17 Tyr-106 and Glu-109) and Val-121 and Glu-126 (RGS17 Ile-143 and Glu-148). A, graph of CSPs for all residues that could be assigned in the ¹H-¹⁵N 2D HSQC spectra of RGS4 (25) upon addition of 40-fold molar excess CaCl₂. B, residues including and adjacent to the putative cation-binding sites in RGS4 that displaced CSPs >2 S.D. greater than the average CSP were used to calculate the K_D,avg for each site by fitting the CSP as a function of ion concentration to a one-site binding model. CSP was determined as a function of increasing Ca²⁺ concentration. The K_D,avg for residues Tyr-84, S85, Glu-86, Glu-87, Asn-88, and Ile-89 is 9.6 ± 3 mm, and the K_D,avg for residues Ala-123, Lys-125, and Val-127 is 6.1 ± 1.6 mm. C, graph of CSPs for all residues assigned in the ¹H-¹⁵N 2D HSQC spectra of RGS4 upon addition of 40-fold molar excess MgCl₂. D, CSP as a function of increasing Mg²⁺ concentration. The K_D,avg for residues Ser-85, Glu-86, Glu-87, and Asn-88 is 93 ± 193 mm, and the K_D,avg for residues Ala-125 and Lys-125 is 90 ± 75 mm.

Figure 8.

CaCl₂ enhances the binding of activated Gα_o by RGS17 but not RGS4. An AlphaScreen assay was using to detect and quantify the binding of RGS17 to Gα_o. A, RGS17 binding to activated Gα_o is increased upon the addition of 5 mm CaCl₂ (purple inverted triangle) relative to the control (blue circle), consistent with Ca²⁺ promoting binding. Addition of 10 mm EGTA, which preferentially chelates free Ca²⁺ in solution, has no effect on the RGS17–Gα_o interaction. In contrast, addition of 10 mm EDTA (red squares), which preferentially chelates free Mg²⁺ in solution, decreases the binding between RGS17 and Gα_o. This could be due to loss of Mg²⁺ bound to RGS17, or it may reflect a decrease in the amount of activated Gα_o, which requires Mg²⁺ for stability. B, quantitation of saturation binding curves shown in A. C, RGS4 binding to activated Gα_o (blue circles) is not altered by CaCl₂ (purple inverted triangles), EDTA (red squares), or EGTA (green triangles). D, quantitation of saturation binding curves shown in C. Data represent the mean of three independent experiments ±S.E. (error bars, *, p < 0.05; **, p < 0.01).

Figure 9.

Ca²⁺ increases RGS17-stimulated GTP hydrolysis. A GTPase-Glo assay was used to detect and quantify RGS-stimulated GTP hydrolysis on a rate-altered Gα_i1 mutant, Gα_i1 R178M/A326S. A, RGS17 increases the rate of GTP hydrolysis in the presence (red squares) or absence (blue circles) of saturating CaCl₂. Addition of CaCl₂ significantly decreased the K_m (Table 2). B, RGS4 stimulates GTP hydrolysis on Gα_i1 but is insensitive to the presence of CaCl₂. Data represents the mean of four independent experiments ±S.E. (error bars).

Figure 10.

ITC characterization of RGS17-Gα_o in the absence (left) and presence (right) of saturating levels of Ca²⁺. K_D values were calculated to be 611 ± 128.5 and 596 ± 257 nm in the absence and presence of Ca²⁺, respectively. The binding enthalpies were −7.33 ± 0.72 and −2.76 ± 0.74 kcal/mol in the absence and presence of Ca²⁺, respectively.