A new approach to producing functional G alpha subunits yields the activated and deactivated structures of G alpha(12/13) proteins

Abstract

The oncogenic G(12/13) subfamily of heterotrimeric G proteins transduces extracellular signals that regulate the actin cytoskeleton, cell cycle progression, and gene transcription. Previously, structural analyses of fully functional G alpha(12/13) subunits have been hindered by insufficient amounts of homogeneous, functional protein. Herein, we report that substitution of the N-terminal helix of G alpha(i1) for the corresponding region of G alpha12 or G alpha13 generated soluble chimeric subunits (G alpha(i/12) and G alpha(i/13)) that could be purified in sufficient amounts for crystallographic studies. Each chimera bound guanine nucleotides, G betagamma subunits, and effector proteins and exhibited GAP responses to p115RhoGEF and leukemia-associated RhoGEF. Like their wild-type counterparts, G alpha(i/13), but not G alpha(i/12), stimulated the activity of p115RhoGEF. Crystal structures of the G alpha(i/12) x GDP x AlF4(-) and G alpha(i/13) x GDP complexes were determined using diffraction data extending to 2.9 and 2.0 A, respectively. These structures reveal not only the native structural features of G alpha12 and G alpha13 subunits, which are expected to be important for their interactions with GPCRs and effectors such as G alpha-regulated RhoGEFs, but also novel conformational changes that are likely coupled to GTP hydrolysis in the G alpha(12/13) class of heterotrimeric G proteins.

Figure 1

Generation of Gα_i/13 and Gα_i/12 chimeric proteins. (A) Schematic representation of the Gα_i/13 and Gα_i/12 chimera. Residues of Gα_i1 are shown in white, and numbers within the colored regions of the Gα_i/13 or Gα_i/12 chimera correspond to original numbering of residues contributed by Gα₁₃ (black) or Gα₁₂ (gray). (B) Purified His₆-Gα_i/13 or His₆-Gα_i/12 (3 µg) were subjected to SDS-PAGE and stained with Coomassie Brilliant Blue. The position and apparent molecular weight of standard proteins (in kDa) is indicated. (C) GTPγS binding to Gα_i/13 (●) or wild-type Gα₁₃ (○) was measured at 30°C. Data are the mean of duplicate determinations. (D) Gα_i/13 or Gα_i/12 functionally interact with Gβγ. GTPγS binding to Gα_i/13 or Gα_i/12 (3 µg each) was measured either in the absence or presence of Gβγ (20 µg) as indicated. GTPγS binding was quantified after 90 min at 30°C. Data are the mean of duplicate determinations.

Figure 2

Gα_i/13 or Gα_i/12 chimera interact with RH domains in an activation-dependent manner. GST-p115-RH or GST-LARG-RH were incubated with purified His₆-Gα_i/13 (upper panels) or His₆-Gα_i/12 (lower panels), either in the absence or presence of AlF₄⁻, and then GST-RH was pulled down using glutathione Sepharose 4B resin. Protein eluted from washed beads was resolved by SDS-PAGE and either immunoblotted (IB) with anti-Gα₁₃ (B860) or anti-Gα₁₂ (J168) antibody, or stained by Coomassie Brilliant Blue (CBB).

Figure 3

Activity of purified Gα_i/13 and Gα_i/12 in reconstitution assays. (A) Single-turnover hydrolysis of GTP bound to Gα_i/13 (upper panel) or Gα_i/12 (lower panel) was measured at 15°C in the absence (□) or presence of 100 nM GST-p115-RH (●) or 100 nM GST-LARG-RH (▲). Data are from one experiment, which is representative of three experiments. (B) Stimulation of p115RhoGEF activity by Gα_i/13 but not Gα_i/12. GTPγS binding to His₆-RhoA was measured after incubation for 5 min at 30°C in the presence of p115RhoGEF (5 nM) and indicated AlF₄⁻-activated Gα subunit (100 nM). Data are the mean of duplicate determinations from one experiment, which is representative of three experiments. (C) Dose-dependent stimulation of p115RhoGEF activity by Gα_i/13. GTPγS binding to His₆-RhoA by p115RhoGEF (5 nM) was evaluated after 5 min at 30°C, and included the indicated concentration of either AlF₄⁻-activated wild-type Gα₁₃ (○) or Gα_i/13 (●). Samples lacking RhoA but containing p115RhoGEF and either AlF₄⁻-activated wild-type Gα₁₃ (△) or Gα_i/13 (▼) were assayed in parallel.

Figure 4

Structures of Gα_12/13 subunits in activated and deactivated states. (A) The activated Gα_i/12·GDP·AlF₄⁻ complex. The region of the αB-αC loop distinct from that of Gα_i/13 is colored purple. The three conformationally flexible “switch regions” of the Gα subunit are red and labeled with Roman numerals (I–III). A fourth, apparently Gα_12/13-specific element (the “spring”) is likewise colored red. The Mg²⁺·GDP·AlF₄⁻ ligand complex is shown as a ball-and-stick model, with carbons colored gray, oxygens red, nitrogens blue, phosphates yellow, fluorines purple, aluminum cyan and magnesium black. Waters are shown as red spheres. (B) The deactivated Gα_i/13·GDP complex adopts an unusually open conformation in which the α-helical domain has rotated ~8.5° away from the Ras-like domain. Switch II is almost completely disordered, and switch III has rotated away (up in the figure) from the nucleotide binding site. (C) Stereo view of the C^α traces of Gα_i/12·GDP·AlF₄⁻ (green with red switch regions) and Gα_i/13·GDP (black with yellow switch regions), superimposed using their Ras-like domains. The glutamate side chain in the “spring” of each α-helical domain (E175 in Gα₁₂, E172 in Gα₁₃) is shown as a stick model.

Figure 5

The active sites and α-helical domains of activated and deactivated Gα_12/13 subunits. (A) The activation-dependent “spring” of the α-helical domain. In the deactivated (GDP-bound) state, Gα_i/13-E172 (yellow) is extended to form a salt bridge with Lys292 and packs against the nucleotide. In the activated (AlF₄⁻-bound) state, the spring (red) adopts a curled conformation and Gα_i/12-Glu175 does not contact the bound nucleotide. Only the Ras-like domain of Gα_i/13 is shown for clarity. Electron density from a 2.5 σ |F_o|-|F_c| omit map from the Gα_i/12·GDP·AlF₄⁻ crystal structure is shown as green wire cage. (B) Superposition of the activated and deactivated conformations of the Gα₁₃ α-helical domain. The C^α-trace of the α-helical domain (residues 74 to 201) and the β6-α5 loop (residues 345 to 357) of the Gα_i/13·GDP structure are colored black, and the α-helical domain from the Gα_13/i-5·p115RhoGEF complex is colored green. The αD-αE1 loop in the α-helical domain and the linker between the Ras-like and α-helical domains (N-terminal to αA) exhibit the most profound conformational changes upon nucleotide hydrolysis. Electron density from a 1.0 σ 2|F_o|-|F_c| map derived from the Gα_i/13·GDP crystal structure is shown as green wire cage.