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J. Biol. Chem., Vol. 282, Issue 3, 1905-1915, January 19, 2007

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Biochemical and Structural Characterization of the Gem GTPase^*

Anne Splingard¹, Julie Ménétrey^¶1, Mylène Perderiset^¶1, Jérome Cicolari^¶, Karine Regazzoni^¶, Fatima Hamoudi^¶, Lucien Cabanié^¶, Ahmed El Marjou^¶, Amber Wells^¶, Anne Houdusse^¶2, and Jean de Gunzburg³

From the Institut Curie, Centre de Recherche, Paris F-75248, France, INSERM U528, Paris F-75248, France, and ^¶CNRS UMR 144, Paris F-75248, France

Received for publication, May 8, 2006 , and in revised form, October 4, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUSION REFERENCES

 
RGK proteins, encompassing Rad, Gem, Rem1, and Rem2, constitute an intriguing branch of the Ras superfamily; their expression is regulated at the transcription level, they exhibit atypical nucleotide binding motifs, and they carry both large N- and C-terminal extensions. Biochemical and structural studies are required to better understand how such proteins function. Here, we report the first structure for a RGK protein: the crystal structure of a truncated form of the human Gem protein (G domain plus the first part of the C-terminal extension) in complex with Mg·GDP at 2.1 Å resolution. It reveals that the G-domain fold and Mg·GDP binding site of Gem are similar to those found for other Ras family GTPases. The first part of the C-terminal extension adopts an -helical conformation that extends along the 5 helix and interacts with the tip of the interswitch. Biochemical studies show that the affinities of Gem for GDP and GTP are considerably lower (micromolar range) compared with H-Ras, independent of the presence or absence of N- and C-terminal extensions, whereas its GTPase activity is higher than that of H-Ras and regulated by both extensions. We show how the bulky DXWEX motif, characteristic of the switch II of RGK proteins, affects the conformation of switch I and the phosphate-binding site. Altogether, our data reveal that Gem is a bona fide GTPase that exhibits striking structural and biochemical features that should impact its regulation and cellular activities.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUSION REFERENCES

 
Gem is an atypical protein of the Ras superfamily that belongs to the recently described RGK (Rad, Kir/Gem) subfamily encompassing the Rad, Gem (and its mouse ortholog Kir), Rem1 (also known as Rem or Ges), and Rem2 proteins (1). These proteins are thought to control the activity of L-type Ca²⁺ channels by interacting with their -subunit (2–4) as well as to intervene in cytoskeletal dynamics. Indeed, Gem interacts with the microtubule network through the kinesin-like protein KIF9 (5) and regulates actin dynamics downstream from RhoA by inhibiting Rock (6) and interacting with the Rho GTPase-activating protein Gmip (7).

GTPases of the RGK family exhibit several specific features in their sequence that distinguish them from other members of the Ras superfamily (see Fig. 1). First, RGK members possess large N- and C-terminal extensions beyond the Ras-homologous G domain. The function of the N-terminal extension of 44–88 residues (71 residues in the case of Gem) is presently unknown. The C-terminal extension of 29–37 residues (31 residues for Gem) is devoid of consensus lipid modification sequences (prenylation or palmitoylation) despite having a conserved cysteine residue at the seventh position from the C terminus, but it carries a functional Ca²⁺/CaM⁴ binding site. There is evidence that the C terminus of Gem is necessary for its interaction with membranes (8) and that its association with Ca²⁺/CaM maintains Gem in the cytosol (9). Last, RGK proteins exhibit substitutions at key positions for GDP/GTP binding and hydrolysis: (i) within the PM1 motif (P-loop, GXXXXGK(S/T)) that is involved in phosphate binding, the residue equivalent to Gly¹² in H-Ras, whose mutation causes the constitutive activation of Ras and Rho family proteins, is changed to glutamine in Gem, proline in Rad and Rem2, and serine in Rem1; (ii) within motifs that belong to the switch I and II regions, which are sensors for the presence of the -phosphate and thus drive the conformational switch of Ras family proteins during the GDP/GTP cycle, the highly conserved threonine residue from the PM2 motif in switch I (Thr³⁵ in H-Ras) is lacking in the RGK family, whereas the conserved PM3 motif (DTAGQ motif) in switch II is changed to a bulky DXWEX motif. Beyond its -phosphate sensor/binding role, the DTAGQ motif is also critical for GTP hydrolysis, due to the important role that the glutamine residue (Gln⁶¹ in H-Ras) plays in the catalytic mechanism. This residue is conserved in Rad and Rem2 but is replaced by an alanine in Rem1 and an asparagine in Gem. Because of these changes, the structural properties of the regions of RGK proteins involved in interacting with nucleotides as well as the biochemical features of GDP/GTP binding and GTP hydrolysis may be quite different from those of other Ras family proteins.

In order to investigate these questions, we have solved the crystal structure of a truncated form of human Gem, encompassing the G domain plus the first part of the C-terminal extension (Met⁷³–Arg²⁶⁴) in complex with Mg·GDP at 2.1 Å resolution, and characterized its biochemical activities. In addition, we have investigated the influence of the N- and C-terminal extensions on the biochemical properties of Gem, including the association of CaM with its C-terminal region.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUSION REFERENCES

 
Bacterial Expression Constructs—Sequences encoding various portions of the human Gem protein were cloned in the bacterial expression vector pGST//1, a derivative of pGEX-4T1 in which the thrombin cleavage site has been replaced by a TEV protease cleavage site (10). To express the portion of Gem devoid of the full N-terminal extension and of the last part of the C-terminal extension (GemNCaM) that we consider as homologous to Ras, sequences encoding residues Met⁷³–Arg²⁶⁴ were amplified by PCR and cloned between the EcoRI and SalI sites of pGST//1, leading after proteolytic cleavage to a protein containing a 7-residue linker (GAMDPEF) upstream from residue Met⁷³. GemNC7 and GemC7, encompassing residues Met⁷³–Ser²⁸⁹ and Met¹–Ser²⁸⁹, respectively, were similarly cloned into NcoI-SalI-digested pGST//1, thus leading to proteins with a 3-residue N-terminal linker (GAM) after TEV protease cleavage. The 5' primers used were 5'-T GTT GCC ATG GGA ACC TAC TAC CGA GTG GTG CTC-3' for Gem NC7 and 5'-T GTT GCC ATG GGA ACT CTG AAT AAT GTC ACC ATG CG-3' for GemC7; in both cases, 5'-GAA TCT GTC GAC C CTA GGA TTT GGA CTT GAG CTT GAA GG-3' was used as a 3' primer. The integrity of cloned sequences was verified by DNA sequencing.

Production and Purification of Gem Proteins—Plasmids were transformed into Escherichia coli BL21, and proteins were produced by induction at 30 °C for 5 h with 0.2 mM isopropyl -D-thiogalactopyranoside. For biochemical purposes, proteins were purified as follows. Frozen bacteria were resuspended in 50 mM Tris-HCl, pH 8, containing 100 mM NaCl, 10 mM MgCl₂, 1 mM EDTA, 10% glycerol, 2 mM DTT, 0.2% Triton X-100, antiprotease mixture (Roche Applied Science), 10 µg/ml aprotinin, 1 mM benzamidine, 1 µg/ml pepstatin. The GemNC7 protein was insoluble unless co-produced with CaM (expressed using a previously described vector) (11); in this case, 2 mM CaCl₂ was added to the lysis buffer and kept during the whole purification procedure. The suspension was incubated with 0.5 mg/ml lysozyme for 30 min at 4 °C and brought to room temperature for 15 min after the addition of 1 µg/ml DNase I, and the lysate was centrifuged at 50,000 x g for 30 min at 4 °C. The supernatant was incubated with glutathione-Sepharose 4B (Amersham Biosciences) for 2 h at 4°C; beads were washed once with 50 mM Tris-HCl, pH 8, 300 mM NaCl, 2 mM DTT, 10 mM MgCl₂ and twice with the same buffer containing only 100 mM NaCl and protease inhibitors. Gem was cleaved from GST with rTEV protease on the beads in the last washing buffer overnight at 4 °C; the effluent was passed through successive Ni²⁺-nitrilotriacetic acid-agarose (Qiagen) and glutathione-Sepharose 4B columns to remove the rTEV protease and GST tag. Gem was concentrated using Centricon tubes (Millipore), snap-frozen in liquid nitrogen, and kept at -80 °C. The purity of the preparations was assessed by SDS-PAGE (supplementary Fig. S1). Protein concentration was measured with a dye-binding assay (Bio-Rad) using BSA as a standard; as determined by [8-³H]GDP binding assays, our purified preparations contained 50–70% active protein. CaM was dissociated from GemNC7 by treatment with 2 mM EGTA for 10 min on ice; CaM was adsorbed onto Q-Sepharose (Amersham Biosciences) by a 15-min incubation at 4 °C and separated from GemNC7 by centrifugation. To form the GemC7·CaM complex, 100 µgof GemC7 was incubated with a 10-fold excess of purified CaM (11), and the complex was purified by gel filtration on a Superdex 75 column (Amersham Biosciences). Alternatively, the GemC7·CaM complex was directly purified from bacteria co-expressing both proteins as described above for the GemNC7·CaM complex followed by gel filtration on a Superdex 75 column.

For crystallization purposes, GemNCaM was purified as follows. Frozen bacteria were resuspended in 50 mM Tris-HCl, pH 8, containing 250 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 0.1 mM GDP, 10% glycerol, 0.5 mg/ml lysozyme, and 5 mM DTT. They were disrupted by sonication, the lysate was ultracentrifuged at 100,000 x g for 30 min at 4 °C, and the supernatant was incubated at 4 °C with glutathione-Sepharose 4B beads for 2 h. The GST fusion protein was eluted by glutathione and cleaved with rTEV protease overnight at 4 °C, and rTEV and GST were removed as above. Gem was further purified by anion exchange chromatography on a MonoQ 5/5 column (Amersham Biosciences), concentrated to 12 mg/ml, frozen to liquid N₂, and stored at -80 °C in 25 mM Tris-HCl, pH 8, 150 mM NaCl, 5 mM MgCl₂, 1 mM GDP, and 1 mM DTT.

Nucleotide Binding and Dissociation—Gem protein in its various forms (0.5 µM) was incubated for increasing amounts of time at 30 °C in binding buffer (20 mM Hepes, pH 7.5, 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 10 mM MgCl₂) containing 50 µg/ml BSA and either 10 µM [8-³H]GDP (11.7 Ci/mmol, Amersham Biosciences; 1:10 isotopic dilution) or 40 µM [5--³⁵S]GTP (1000 Ci/mmol, Amersham Biosciences; 1000 cpm/pmol) in a total volume of 250 µl. 10-µl portions were removed at the indicated times, diluted in 2 ml of cold binding buffer, filtered through nitrocellulose filters (Schleicher and Schuell NC45; pore size 0.45 µm), and washed three times with 2 ml of the same buffer. Radioactivity on the filter corresponding to protein-bound nucleotide was determined by liquid scintillation counting. Measures were performed in duplicate or triplicate. To determine kinetics of nucleotide dissociation, a 1 mM excess of unlabeled GDP or GTP was added to the binding reactions described above after 2 h of nucleotide binding. 10-µl portions were removed at the indicated times and treated as above to measure the amount of radiolabeled nucleotide remaining bound to the protein. The affinity of Gem for nucleotides was determined by incubating 4 pmol of Gem for 2 h at 30 °C in binding buffer with 0–30 µM [8-³H]GDP or 0–300 µM [5--³⁵S]GTP; protein-bound nucleotide was measured as above. The competition of various nucleotides for GDP binding to Gem was assessed by measuring the binding of 10 µM [8-³H]GDP to 0.5 µM Gem in the presence of 0–100 mM unlabeled competitors (GMP, GDP, GTP, GTPS, GMP-PNP). GTP was repurified by ion exchange chromatography (Mono Q); all GTP analogues (labeled and unlabeled) were freed from contaminating GDP by treatment with agarose-bound alkaline phosphatase (Sigma) immediately prior to use. Contamination of labeled GTP³⁵SbyGDP³⁵S was assessed to be less than 0.03% by thin layer chromatography on polyethyleneimine-cellulose.

GTPase Activity—Gem proteins (0.5 µM) were incubated with 20 µM [-³²P]GTP (3000 Ci/mmol; Amersham Biosciences; 4000 cpm/pmol) in 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM DTT, 10 mM MgCl₂ at 30 °C. 1-µl portions were removed at the indicated times and immediately mixed at 4 °C with 2 µl of a solution containing 0.2% SDS, 5 mM EDTA, 5 mM GDP, and 5 mM GTP. Samples were heated to 70 °C for 2 min to dissociate protein-bound nucleotides, and 1-µl aliquots were spotted onto polyethyleneimine-cellulose-covered thin layer chromatography plates (Merck). They were developed in 0.6 M sodium phosphate buffer, pH 3.4, for 30 min, dried, and autoradiographed. The amounts of labeled nucleotides were measured on the plates using a Storm 860 PhosphorImager (Amersham Biosciences), and the relative proportion of hydrolyzed GTP was calculated using ImageQuant.

Crystallization and Structure Determination—Crystallization assays were performed with the NCaM portion of Gem (residues Met⁷³–Arg²⁶⁴) using the hanging drop vapor diffusion method at 16 °C. Crystals were grown in a solution containing 0.9–1.1 M sodium acetate, 100 mM sodium cacodylate, pH 6.5, 5 mM MgCl₂, and 2 mM DTT. Crystals appeared by self-nucleation in 1–2 h and reached their final dimension within 48 h of mixing equal volumes of protein (10 mg/ml) and reservoir solution. Crystals were frozen in liquid nitrogen using the crystallization solution containing 1.4 M sodium acetate and 20% glycerol as cryoprotectant. The first data set was collected on a laboratory source (IBPC, Paris, France) at 3.6 Å and allowed to solve the structure by molecular replacement. Subsequently, diffraction data up to 2.1 Å were collected at -170 °C on beam line BM14 of the European Synchrotron Radiation Facility. Intensities were integrated with MOSFLM (12) and scaled with SCALA (12). Crystals of Gem-GDP belong to the primitive hexagonal space group P6₁ with a = b = 116.6 Å, c = 81.4 Å. The asymmetric unit contains two molecules (A and B) with a high V_m of 3.65 ų/Da and a solvent content of 66.3%. Molecular replacement was performed with AmoRe (13) using as search model Rap2A-GDP (14) (Protein Data Bank code 1KAO [PDB] ). The model included only the protein backbone chain, omitting the switch regions and the Mg·GDP ligand. The electron density clearly indicated the presence of Mg·GDP in the active site, which was modeled before starting refinement.

Structure Refinement—The structure was refined by maximum likelihood refinement with CNS (15) and Refmac (12) and by graphical building using TURBO (16). ARP-wARP was used for automatic building of water molecules (17). The structure consists of two Gem molecules of 351 residues, two Mg·GDP, 127 waters, and seven arsenic atoms. Some side chains are poorly defined in the current structure and were modeled with null occupancy, making no contact with vicinal residues. Note that in molecule B, three N-terminal residues (Glu, Phe, and Gly) resulting from the TEV cleavage site of the GST fusion tag were modeled in electron density and numbered as residues 70–72. Regions Met¹⁰²–Asp¹⁰⁵ and Lys¹³⁶–Asn¹³⁹ from molecule A and His⁹⁹–Gly¹¹⁰ and Lys¹³⁶–Leu¹⁴² from molecule B have no defined electron density and were omitted from the model. Several exposed cysteine side chains (Cys¹⁰⁶, Cys¹⁹⁸, Cys²⁰⁹, and Cys²⁰⁵ in molecule A and Cys¹⁴⁶, Cys¹⁹⁸, and Cys²⁰⁹ in molecule B) were partially modified to S-dimethylarsenic cysteine by the cacodylate buffer. The presence of arsenic was confirmed by anomalous diffraction data (not shown). Crystallization assays to overcome this chemical modification were unsuccessful. The difference in cysteine modification residues between molecules A and B and the fast crystallization rate suggest that modifications take place following crystallization. Thus, structural differences observed between molecules A and B were considered to be the consequence of different crystal packing contacts. The stereochemistry of the final refined model is excellent, and there is no main - outside allowed regions of the Ramachandran plot. The refined structure has a crystallographic R-value of 0.209 and a free R-value of 0.241. Crystallographic statistics are summarized in Table 2. Figures were produced using Molscript (18) and Raster3D (19).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUSION REFERENCES

Nucleotide Binding and Dissociation—As is the case with most small GTPases, it is likely that purified Gem contains a stoichiometric amount of bound nucleotide. Hence, we measured the exchange of this endogenous nucleotide for exogenously added [³H]GDP or GTP³⁵S and maintained a large excess of exogenous nucleotide relative to protein in order to minimize the possible interference from the endogenous nucleotide released during the exchange reaction. As shown in Fig. 2A, GemNCaM exchanged its endogenous nucleotide for [³H]GDP and GTP³⁵S with similar saturable kinetics. In both cases, binding of exogenous nucleotide was strictly dependent on the presence of high concentrations of free Mg²⁺, with maximal binding reached above 5 mM (not shown). The competition experiment depicted in Fig. 2B shows that GemNCaM bound GDP with a higher affinity than GTP; among GTP analogues, GTPS was almost as effective as GTP, whereas GMP-PNP exhibited a somewhat lower affinity. As is the case with other G proteins, GMP did not compete with GDP for binding to Gem. Binding of labeled GDP or GTPStoGemNCaM was saturable as a function of nucleotide concentration (Fig. 2, C and D, respectively) exhibiting affinities in the micromolar range, with a K_d for GDP of 2.5 µM and a K_d for GTPSof19 µM (Fig. 2E and Table 1), values that are several orders of magnitude larger than those reported for H-Ras (0.02 nM for GDP and 0.005 nM for GTP) (20). These values did not change when a 10-fold lower protein concentration was used in the assay, showing that they actually reflect the affinity of Gem for the nucleotides and do not result from an artifact due to the unlabeled nucleotide remaining bound to Gem throughout its purification (21). Both GDP and GTP dissociated from GemNCaM with single site kinetics (Fig. 2F); as expected from their differential affinities, GTP dissociated significantly faster from GemNCaM than GDP with half-times of 50 s and 8 min, respectively (Table 1). These values are 1 (for GDP) to 2 (for GTP) orders of magnitude smaller than for H-Ras, from which both GDP and GTP exhibit dissociation half-times of 45 min (20).

View larger version (62K): [in this window] [in a new window]   FIGURE 1. Sequence alignment for RGK proteins. Conserved residues are indicated by dots, conserved motifs are highlighted in gray, and their names are given below. *, position of documented phosphorylation sites. Note that multiple conserved serines are found in the N- and C-terminal extensions. A schematic diagram of the secondary structures of Gem is shown above the sequence. The sequence of H-Ras is aligned along those of RGK proteins for comparison. SH3, Src homology 3.

GTPase Activity—Because of the important changes in the switch II region between Gem and H-Ras, assessing whether Gem carries an intrinsic GTPase activity was of particular interest. As shown in Fig. 3, GemNCaM indeed hydrolyzed GTP to GDP as assessed by thin layer chromatography. That this reaction genuinely represented the GTPase activity of Gem, and not that of a contaminant phosphatase, was attested by the fact that ³²P-labeled GDP accumulated as a function of time and that no ³²P-labeled GMP was found among reaction products (not shown). In the case of H-Ras and most related GTPases, only a single cycle of intrinsic GTP hydrolysis can be measured for each molecule in the absence of guanine nucleotide exchange factors and GTPase-activating proteins, because the kinetics of product (GDP) dissociation are slower than the rate of hydrolysis, thereby preventing binding of a new substrate (GTP) molecule to the enzyme. In contrast, the rate of GDP dissociation from Gem is sufficiently rapid as compared with the rate of GTP hydrolysis, so that enzymatic activity representing several cycles of hydrolysis was measured for Gem. Nevertheless, rate constants for the initial velocity of the hydrolysis reaction may be compared; the k_cat of the reaction measured for GemNCaM was 30-fold higher than that of H-Ras (Table 1).

View larger version (25K): [in this window] [in a new window]   FIGURE 2. Biochemical characteristics of GemNCaM. A, nucleotide binding kinetics. 0.5 µM protein was incubated for the indicated time intervals with 10 µM [3H]GDP () or 40 µM GTP35S (), and bound nucleotide was measured as detailed under "Experimental Procedures." B, competition of various nucleotides for GDP binding to Gem. , GDP; •, GTP; , GTPS; , GMP-PNP; , GMP. C, binding of [3H]GDP to Gem as a function of GDP concentration (). D, binding of GTP35S to Gem as a function of GTPS concentration (). E, Scatchard plot of the data from C () and D (). F, dissociation of nucleotides from Gem. Dissociation of previously bound [3H]GDP by excess unlabeled GDP () and previously bound GTP35S by unlabeled GTPS () are shown.

Overall Structure of Gem and Its Nucleotide Binding Site—The crystal structure of GemNCaM was determined in complex with Mg·GDP at 2.1 Å resolution (Table 2 and Fig. 4A). Two molecules (A and B) of Gem are present in the asymmetric unit and are virtually identical, yielding a root mean square deviation value of 0.6 Å (on 158 C excluding the switch regions). The overall structure of the G domain of Gem resembles that of other small GTPases with the classical six-stranded -sheet, surrounded by five -helices (a root mean square deviation value of 0.79 Å on 132 C is obtained when compared with H-Ras-GDP (Protein Data Bank code 4Q21 [PDB] ) (22). In addition to the G domain, the structure reveals that the first part of the C-terminal extension (Asp²⁴⁴–Arg²⁶⁴) (Fig. 1) folds into an helix, which contacts the tip of the interswitch (strands 2-3 between switch I and switch II) and extends antiparallel along the 5 helix (Fig. 4A). The electron density maps unambiguously show the presence of a tightly bound GDP molecule and a Mg²⁺ ion at the active site (supplemental Fig. S2). The bound nucleotide has extensive hydrophobic and hydrophilic interactions with the surrounding residues (Fig. 4B and supplementary Table S1) that are conserved in other small GTPases. In the GemNCaM-GDP structure, the Mg²⁺ ion is coordinated in a classical manner by six oxygens in a virtually perfect octahedral geometry with the hydroxyl group of the P-loop Ser⁸⁹ O (2.2 Å), the -phosphate of GDP (2.0 Å), and four water molecules (2.0–2.3 Å) (Fig. 4B).

View larger version (32K): [in this window] [in a new window]   FIGURE 3. GTPase activity of Gem. A, the various forms of Gem (0.5 µM) were incubated with 20 µM [-32P]GTP, and reaction products were separated by thin layer chromatography on polyethyleneimine-cellulose-covered plates. B, quantification of the data in A. , GemC7; , GemNC7-CaM; , GemNCaM.

The Switch Regions—Two distinct conformations are observed for the switch regions in molecules A and B with Gly⁹⁷/Asp¹¹² and Trp¹³³/Val¹⁴⁹ as hinge residues for switch I and II, respectively (Fig. 4A). Note that only the ends of both switches have interpretable electron densities and were modeled, whereas regions Met¹⁰²–Asp¹⁰⁵ and Lys¹³⁶–Asn¹³⁹ for molecule A and regions His⁹⁹–Gly¹¹⁰ and Lys¹³⁶–Lys¹⁴² for molecule B were omitted from the model, respectively, for switch I and II. The distinct conformations of both switch extremities are driven by crystal packing contacts. This suggests that in the absence of protein contacts, the switch regions are disorganized in the GDP-bound form of GemNCaM. However, in the absence of a structure for the GDP-bound form of full-length Gem, it is not possible to anticipate how the N- and C-terminal extensions will impact the conformation of the switch regions.

View larger version (61K): [in this window] [in a new window]   FIGURE 4. Crystal structure of GDP-bound Gem-NCaM. A, an overall view of Gem-NCaM-GDP is shown with switch I in green, the interswitch in yellow, switch II in pink, and the C-terminal helix in blue. Note that both conformations of switch I and II are shown superposed in dark for molecule A and in bright for molecule B. B, the scheme of Mg·GDP interactions is given for molecule A with distances (see supplemental Table S1 for distances in molecule B). An inset of the magnesium coordination sphere is shown separately for clarity. C, close view of the C-terminal helix (blue) and its interaction with the interswitch (yellow) and the 5 helix (white) with secondary structure shown as coils. Hydrogen bonds are indicated by dashed lines. Note that for clarity, the orientation is not strictly conserved with the overall view (A). D, close view of the DMWEN motif in switch II (pink). The side chain of Glu134 from molecule A is shown as transparent, indicating its partial occupancy. Orientation with the overall view is conserved.

View larger version (50K): [in this window] [in a new window]   FIGURE 5. Comparison of the switch regions of GemNCaM-GDP with H-Ras-GDP/GTP. A, the H-Ras-GDP (Protein Data Bank code 4Q21; transparent light blue) and H-Ras-GTP (Protein Data Bank code 5P21; transparent dark blue) switch I-interswitch-switch II regions are shown superposed on the GemNCaM-GDP structure (same colors as in Fig. 4) for comparison. B, close view of A with overall structure shown as coils and the C-terminal part of switch II deleted for clarity. The first residues from the DMWEN and DTAGQ motifs in Gem and H-Ras, respectively, are shown in ball-and-stick representations for comparison. Note the divergent backbone trace and side chain directions between Gem and H-Ras after the Asp131/Asp57 residues and the position of Met132 that shortens the 2 strand, pushing switch I away from the nucleotide binding site in Gem when compared with H-Ras.

Structural comparison of the DMWEN motif in Gem-GDP with the DTAGQ motif in H-Ras-GDP/GTP (22, 23) (Fig. 5) reveals that the bulky tryptophan (Trp¹³³ in Gem) that is conserved in all RGK proteins and replaces an alanine residue in other small GTPases imposes a unique conformation on this region (Fig. 5A). Its bulky side chain is turned outward from the nucleotide binding site packing along a hydrophobic patch composed of the C terminus of the 1 strand (Ile⁸¹-Gly⁸²), the 3 helix (Leu¹⁷¹), and switch II (Met¹⁴⁷ in both molecules and locked by His¹⁴⁵ in molecule A). This differs from the equivalent alanine residue (Ala⁵⁹)ofthe DTAGQ motif that extends in the direction of the nucleotide-binding site (Fig. 5B). Such a position for Trp¹³³ forces the backbone of the preceding residue (Met¹³²) to protrude toward the N terminus of the 2 strand at 2.3 Å (C atoms) from the position found for the equivalent threonine residue (Thr⁵⁸) in H-Ras. A consequence of this protrusion is that Asp¹¹² makes a bend to avoid a steric clash with the Met¹³² backbone, thus shortening the 2 strand by 1 residue at its N terminus when compared with H-Ras (Fig. 5).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUSION REFERENCES

 
Structural comparisons between the G-domain of Gem and those of other Ras superfamily GTPases reveal that Gem adopts their conserved fold and GDP binding site. Accordingly, Gem also binds GDP and GTP and exhibits an intrinsic GTPase activity. Hence, Gem exhibits the hallmarks of a bona fide GTPase of the Ras superfamily. These structural properties should be shared among all proteins of the RGK family, consistent with the rather high sequence conservation within their G domains (48–64% identity versus 27.5% identity with the G domain of H-Ras). Nevertheless, our study shows that some striking structural elements distinguish Gem from other Ras family proteins, which may be responsible for its distinctive biochemical properties.

The C-terminal Extension—The first part of the C-terminal extension of Gem (Asp²⁴⁴–Arg²⁶⁴; Fig. 1), in the absence of the rest of this region, folds into an -helix (called here Cter) that packs at the tip of the interswitch lining antiparallel to the 5 helix (Fig. 4, A and C). The position of this helix relative to the G domain is reminiscent of that of the N-terminal helix in Arf proteins in their GDP-bound form, except that in Arf proteins, the interswitch is retracted, the N-terminal helix lies parallel to the 5 helix, and its interactions with the G domain are mainly hydrophobic (supplemental Fig. S3A). However, despite some structural similarities, no data are available to envision that the C-terminal helix of Gem could be released from the G domain and move away as is observed for the N-terminal helix of Arf proteins during their GDP/GTP cycle (for a review, see Ref. 24). Although the last part of the C-terminal extension containing the CaM-binding site and the C7 motif (seven C-terminal residues; see Fig. 1) is conserved in sequence among proteins of the RGK family, the first part of the C-terminal extension (Cter of Gem) is not (Fig. 1). This region in Rad, which shares 60% sequence identity with the homologous domain of Gem, should also exhibit an -helical conformation. However, Rem1 and Rem2, respectively, possess one and two consensus Src homology 3-binding motifs (PXXP) within this region (Fig. 1) and therefore cannot adopt an -helical conformation. Whether Rem1/Rem2 are actually able to bind Src homology 3 domain proteins through this region has not yet been reported. Thus, it appears that the first part of the C-terminal extension in the RGK family is the site of structural and probably functional distinction between the Gem/Rad and Rem1/Rem2 subgroups. Note that the first part of the C-terminal extension of Ran, which is unrelated in sequence to that of Rem1/Rem2, also is a proline-rich region. In Ran, this region adopts a nonstructured polypeptide stretch that also binds into the groove formed between the tip of the interswitch and the 5 helix (25) (supplemental Fig. S3B). Altogether, these observations highlight that the interface formed by the tip of the interswitch and the 5 helix is a site with which the N- or C-terminal extensions of small GTPases frequently interact.

Is the Switch I of Gem a Classical Switch Region?—In G proteins, the switch regions undergo drastic conformational changes upon the GDP/GTP cycle. To strictly delineate these regions for a given G protein, structures of its GDP- and GTP-bound forms are required. Here, despite the lack of structural data for the GTP-bound form, we have delineated the putative switch regions of Gem based on the distinct conformations observed in molecules A and B as residues Gly⁹⁸–Asp¹¹² and Trp¹³³–Val¹⁴⁹ for switch I and II, respectively. These distinct conformations, which are induced and stabilized by different crystal packing contacts, carry interesting information concerning the structural features of the switch regions. They also underline that in absence of crystal packing contacts, the switch regions of Gem-GDP (in the absence of the full N- and C-terminal extensions) are flexible, except for the C-terminal part of switch II (His¹⁴⁵–Val¹⁴⁹). Note that in the absence of a three-dimensional structure for the full-length Gem protein, we cannot anticipate how these two extensions as well as CaM binding will impact the switch I and II conformations of the GDP-bound form.

However, independent of the presence or absence of the N- and C-terminal extensions, the bulky DXWEX motif exerts a striking structural impact on the conformation of switch I within the G domain of Gem. Indeed, as compared with the structures of other small GTPases, Gem exhibits a 2 strand that is shortened at its N terminus, with residue Asp¹¹² making a bend that opens the conformation of switch I far away from the nucleotide binding site and switch II (Fig. 4A). Arf and Ran GTPases in their GDP-bound forms also exhibit an open switch I conformation, but the N terminus of their 2 strand remains close to the nucleotide-binding site and to switch II (26–30) (supplemental Fig. S3, C–F). The bend at Asp¹¹² in Gem is induced by the presence of the bulky DXWEX motif, whose conformation diverges from that of the classical DTAGQ motif in order to accommodate the bulky tryptophan residue (Fig. 5B). This residue is turned away from the nucleotide-binding site and packed against a hydrophobic patch. The main chain trace of the preceding residue, Met¹³², is forced to make a kink toward the 2 strand, which is therefore prevented from starting at the same position as observed in H-Ras (Fig. 5). Since the tryptophan residue from the DXWEX motif is conserved in RGK proteins and the main chain trace of the preceding residue is sufficient to drive the bend and thus shorten the N terminus of the 2 strand, we expect this striking feature to be shared by all RGK proteins in their GDP-bound form.

Is this position of switch I, far away from the nucleotide binding site, conserved during the GDP/GTP cycle? If such were the case, switch I may not play a role in the GDP/GTP cycle conformational change. This hypothesis is supported by the fact that the strictly conserved threonine residue of the PM2 motif (Thr³⁵ in H-Ras) (Fig. 1), which binds the -phosphate of GTP thereby driving the switch I conformational change during the GDP/GTP cycle, is absent in RGK proteins. This suggests that switch I may have lost its ability to bind GTP and thus to switch during the GDP/GTP cycle. An additional consequence of a switch I open conformation along the GDP/GTP cycle could be that the nucleotide binding site may become accessible to the N- and/or C-terminal extensions, which may thereby exert an effect on the GTPase activity of the protein (see below).

Structural Insights for the Weak Nucleotide Binding Affinity— Gem exhibits affinities for GDP and GTP in the micromolar range, several orders of magnitude weaker than those measured for H-Ras (see Table 1). However, from a physiological point of view, this should not prevent Gem from being constitutively bound to nucleotides in cells, since the average intracellular concentrations of GDP and GTP are thought to be 100 µM and 1 mM, respectively. The weak nucleotide affinity measured for Gem, relative to H-Ras, is not the consequence of atypical interactions with Mg·GDP, since all of the interactions observed in other Ras superfamily GTPases are conserved. The biochemical data presented here reveal that the presence or absence of the N- and C-terminal extensions and CaM do not affect the affinity of Gem for nucleotides, implying that the structural differences that may explain the weaker nucleotide binding properties of Gem are contained within its G domain alone.

RGK proteins do not possess the G₁ motif, consisting of a phenylalanine (equivalent to Phe²⁸ in H-Ras; Fig. 1) that caps the guanine ring and thus contributes to the high affinity of the nucleotide for the binding site. Indeed, it was shown that mutating this residue to leucine in H-Ras and Cdc42 greatly enhances their nucleotide dissociation rates, without affecting their nucleotide association kinetics or GTPase activity (31, 32). At this position in Gem, an alanine residue, Ala²²³ from the G3 motif (Figs. 1 and 4B), is found above the guanine ring. This shorter residue cannot substitute for the phenylalanine. This sequence difference probably contributes to the relatively weak nucleotide affinity and rapid nucleotide dissociation kinetics observed for Gem. Although Gem, Rad, and Rem2 possess an alanine residue in this position, Rem1 has a threonine residue, which suggests that it may have a slightly higher affinity for nucleotides than other RGK proteins. A striking observation in our structure is that in molecule A, a phenylalanine residue from a crystal symmetry molecule caps the guanine ring in a position similar to that of the phenylalanine G1 motif in other small GTPases (supplemental Fig. S4). This additional interaction in molecule A appears to be responsible for the difference in temperature factors observed between Mg·GDP from molecule A and molecule B (Table 2), thereby supporting the hypothesis that in the presence of such an interaction, Mg·GDP would have a higher binding affinity.

We anticipate that the presence of the atypical DXWEX motif at the beginning of switch II, close to the position of the -phosphate, also reduces the affinity of RGK proteins for nucleotides. First, and as discussed above, the DXWEX motif with the bulky Trp¹³³ imposes a bend at the beginning of the 2 strand that directs switch I away from the nucleotide-binding site. Thus, switch I in this open conformation cannot cover the nucleotide, a feature that should contribute to the weaker measured nucleotide affinity. Second (and also due to the bulky Trp¹³³), the carbonyl main chain of Met¹³², the residue of Gem equivalent to the threonine in the DTAGQ motif, can no longer make a hydrogen bond with the water molecule that binds the magnesium ion and Asp¹³¹ (Figs. 4D and 5B). Thus, the magnesium ion coordination lacks a water-mediated contact in the GDP-bound form (probably also in the GTP-bound form), which should further reduce the nucleotide affinity. Finally, the presence of a glutamate residue in the DXWEX motif close to the phosphate binding site, conserved in all RGK proteins, should contribute to diminish the nucleotide affinity by approaching its negatively charged side chain close to the - and -phosphates inducing repulsion forces. This should be even more pronounced for the GTP-bound form of the protein, since the carboxylate side chain group of Glu¹³⁴ will partially overlap with the -phosphate (supplemental Fig. S5), which may explain the faster dissociation kinetics and lower affinity of Gem for GTP as compared with GDP.

An Unexpected High GTPase Activity—The following biochemical observations came quite unexpected. (i) Gem exhibits an intrinsic GTPase activity, (ii) it is high relative to H-Ras (see Table 1), and (iii) it is greatly affected by the presence or absence of the N- and C-terminal extensions. The first observation is intriguing if one considers that Gem does not exhibit the consensus DTAGQ motif that is critical for GTP binding and hydrolysis in other small GTPases. In addition, the N- and C-terminal extensions of Gem are required for full GTPase activity but are not essential for the G domain of Gem to exhibit a GTPase activity. Altogether, these observations suggest that the molecular mechanism of the intrinsic GTPase activity of Gem is probably different from that of other small GTPases.

The GTPase activity of Gem is reduced when both the N- and C-terminal extensions are deleted and is severely impaired when the N terminus alone is deleted. Note that Ca²⁺/CaM has no effect on the GTPase and GTP binding activities of Gem. This discrepancy with an earlier report (33) may be related to the fact that we took care to remove the N-terminal GST tag from Gem during its purification. However, this raises an intriguing question concerning the observed inhibition of the GTPase activity by the C-terminal extension as to how this inhibition can take place without being affected by the presence or absence of CaM, which should cover a large part of the C-terminal extension and thus impact its structural properties. One attractive hypothesis is that the C-terminal extension folds back onto the nucleotide-binding site that is uncovered by the open conformation of switch I and interferes by steric hindrance with the GTPase machinery. Such a nonspecific interaction would act to inhibit the GTPase activity regardless of the presence or absence of CaM. In full-length Gem, the N-terminal extension may somehow interact with the C-terminal extension and abolish its inhibitory effect by affecting, for instance, the position of the C-terminal extension relative to the nucleotide-binding site. Such an interaction between the N- and C-terminal extensions could also be responsible for the 30-fold higher GTPase activity of GemC7 as compared with that of GemNCaM by positioning the C-terminal extension favorably for the GTPase machinery. Furthermore, binding of cellular partners to the N-terminal extension could release the C-terminal extension and thereby enable it to fold back onto the nucleotide binding site to inhibit the GTPase activity of Gem. This would represent a novel mechanism for regulating the GTPase activity of a Ras superfamily protein.

	CONCLUSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUSION REFERENCES

 
In the absence of the conserved PM2 (Thr³⁵ in H-Ras) and PM3 (DTAGQ) motifs, it was first thought that RGK proteins would not bind GTP and therefore exhibit no GTPase activity. We show here that Gem binds both GDP and GTP with micromolar affinities and exhibits a high GTPase activity relative to H-Ras. Furthermore, we show that the N- and C-terminal extensions of Gem regulate its GTPase activity. We therefore anticipate that the GDP/GTP conformational cycle and the GTPase reaction of Gem will present important differences from those of other small GTPases. To fully understand the molecular mechanisms that govern Gem, additional structures of the protein bound to GTP in the presence of full N- and C-terminal extensions and also complexed with CaM will be required.

Addendum—Ugochukwu (E. Ugochuk, M. Soundararajan, J. Elkins, C. Gileadi, G. Schoch, F. Sobott, O. Federov, J. Bray, N. Pantic, G. Berridge, N. Burgess, W. H. Lee, A. Turnball, M. Sundstrom, C. Arrowsmith, J. Weigelt, A. Edwards, F. von Delft, and D. Doyle (Structural Genomics Consortium (SGC)), unpublished results) have determined the structure of the G domain of Gem bound to GDP (Protein Data Bank code 2G3Y). In contrast with our structure, their structure lacks the first part of the C-terminal extension. No major difference in the overall structures was observed. However, a striking observation is that Glu¹³⁴ from the DMWEN motif, which points inward toward the nucleotide binding site, is much closer to the magnesium ion and -phosphate position than our structure revealed. This position is similar to the one that we have modeled (supplementary Fig. S5), and, as anticipated, this position perturbs the nucleotide binding site, since no magnesium ion is observed in the site.

	FOOTNOTES

 
The atomic coordinates and structure factors (code 2CJW) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by Association pour la Recherche contre le Cancer Grant 3564 (to A. H.) and Grants 4754 and 3515 (to J. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1 and Figs. S1–S5.

¹ These authors contributed equally to this work.

² To whom correspondence may be addressed: CNRS UMR144, Institut Curie, 26 Rue d'Ulm, 75248 Paris Cedex 05, France. Tel.: 33-1-42-34-63-95; Fax: 33-1-42-34-63-82; E-mail: anne.houdusse{at}curie.fr. ³ To whom correspondence may be addressed: INSERM U528, Institut Curie, 26 rue d'Ulm, 75248 Paris Cedex 05, France. Tel.: 33-1-42-34-66-49; Fax: 33-1-42-34-66-50; E-mail: gunzburg{at}curie.fr.

⁴ The abbreviations used are: CaM, calmodulin; TEV, tobacco etch virus; rTEV, recombinant His₆-tagged TEV protease; GMP-PNP, guanylyl-5'-[,-imido]-triphosphate; GTPS, guanosine 5'-O-(3-thiotriphosphate); Cter helix, C-terminal helix of GemNCaM; GST, glutathione S-transferase.

	ACKNOWLEDGMENTS

 
We thank Pierre Legrand for expert technical help for anomalous diffraction data collection at the European Synchrotron Radiation Facility. We also thank the mass spectrometry staff at the Institut Curie for expert technical help. We thank the Institut de Biologie Physico-Chimique (Paris, France) and Ines Gallay for x-ray generator utilization.

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