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Genes to Cells (2006) 11, 961-968. doi:10.1111/j.1365-2443.2006.00994.x
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Crystal structure of human Rad GTPase of the RGK-family

Arry Yanuar1, Shigeru Sakurai1, Ken Kitano1 and Toshio Hakoshima1,2,*

1 Structural Biology Laboratory, Nara Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara 630-0192, Japan
2 CREST, Japan Science and Technology Agency, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Rad (Ras associated with diabetes) is an RGK-family small GTPase that is over-expressed in the skeletal muscle of humans with type II diabetes. Unlike other small GTPases, RGK family members including Rad lack several conserved residues in the GTPase domain. Here, we report the crystal structure of the GTPase domain of human Rad in the GDP-bound form at 1.8 Å resolution. The structure revealed unexpected disordered structures of both switches I and II. We showed that the conformational flexibility of both switches is caused by non-conservative substitutions in the G2 and G3 motifs forming the switch cores together with other substitutions in the structural elements interacting with the switches. Glycine-rich sequences of the switches would also contribute to the flexibility. Switch I lacks the conserved phenylalanine that makes non-polar interactions with the guanine base in H-Ras. Instead, water-mediated hydrogen bonding interactions were observed in Rad. The GDP molecule is located at the same position as in H-Ras and adopts a similar conformation as that bound in H-Ras. This similarity seems to be endowed by the conserved hydrogen bonding interactions with the guanine base-recognition loops and the magnesium ion that has a typical octahedral coordination shell identical to that in H-Ras.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Rad (Ras associated with diabetes), a small GTPase identified by subtractive cloning, is over-expressed in skeletal muscle of patients with type 2 diabetes mellitus (Reynet & Kahn 1993). Rad inhibits insulin-stimulated glucose uptake in myocyte and adipocyte cell lines (Moyers et al. 1996). Rad and its closely related GTPases, Gem, Kir, Rem and Rem2, form an RGK subfamily in the Ras-family small GTPases (Olson 2002). These RGK-family members play three other important roles in cell biology (Kelly 2005). RGK GTPases function as potent inhibitors of voltage-dependent calcium channels (VDCCs) by direct binding to the ß-subunit of VDCC (Beguin et al. 2001). Moreover, two members of the family, Gem and Rad, modulate Rho-dependent remodeling of the cytoskeleton by directly binding to Rho-kinases, leading to inhibition of kinase activity (Ward et al. 2002). Recently, a putative tumor suppressor, nm23-H1, was reported to convert Rad-GDP to Rad-GTP, in a manner akin to guanine-nucleotide exchange factors (GEFs) (Zhu et al. 1999), and regulate the growth and tumorigenicity of breast cancer (Tseng et al. 2001). Thus, Rad is a key GTPase that is involved in biologically and medically important processes.

Small GTPases possess a common GTPase domain that consisits of about 170 residues with five conserved amino acid motifs, G1–G5, which are involved in GTP binding and hydrolysis and base recognition (Vetter & Wittinghofer 2001). The domain exists in two conformational states and behaves as a binary switch that cycles between the active GTP-binding and the inactive GDP-binding forms. The switch function is achieved by large conformation changes in two flexible regions referred to as switches I and II. Compared with other small GTPases, Rad has distinct features in the amino acid sequence, which might affect GTP binding or GTPase activity. The Rad GTPase core domain, which exhibits ~35% identity with other small GTPases such as H-Ras, contains crucial substitutions in the conserved motifs, G1-G3 and G4 (Zhu et al. 1995). In fact, recombinant Gem, Rad and Rem are known to display exceedingly low levels of intrinsic GTPase activity (Cohen et al. 1994; Zhu et al. 1995; Finlin et al. 2003). Another notable characteristic of Rad is its N- and C-terminal extensions that consist of 85 and 50 residues, respectively. These extensions contain 14-3-3 and calmodulin binding sites (Moyers et al. 1997; Beguin et al. 2006). The C-terminal extension lacks the typical CAAX prenylation motifs needed for post-translational modifications (Takai et al. 2001). In an effort to better understand the structure-function relationship of these novel GTPases, we have determined the first crystal structure of a member of the RGK-family GTPases, human Rad GTPase in complex with GDP at 1.8 Å resolution.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Overall structure

Our crystal contained two Rad molecules, molecules A and B, in the crystallographic asymmetric unit of a P21 crystal lattice. These two molecules form a dimer in the crystal lattice, though Rad exists as a monomer in solution, confirmed by gel filtration giving a single peak at 19 kDa (data not shown). The structure of these two molecules is essentially the same with a small root mean square (r.m.s) deviation of 0.30 Å. Rad contains five {alpha}-helices (A1–A5) and one large ß-sheet that comprises two extended anti-parallel ß-strands (B2 and B3) and five extended parallel ß-strands (B3, B1, B4–B6) (Fig. 1A). These major features of the Rad GTPase domain fold are basically conserved as found in H-Ras and other related small GTPases, consisting of a six-stranded ß-sheet surrounded by helices connected with loops. The bound GDP molecule was found at the canonical nucleotide-binding site (Fig. 1B). The current model contains three poorly defined loop regions, switches I and II and loop A3–B5, which were omitted from the model (Fig. 1A,C). Excluding residues of poorly defined loops and altered helix A2, the C{alpha}-carbon atoms of Rad when superimposed on the GDP-bound forms of H-Ras (Tong et al. 1991) and RalA (Nicely et al. 2004) yield relatively small r.m.s. deviations of 0.79 Å and 0.86 Å for 124 and 128 C{alpha}-carbon atoms, respectively (Fig. 2A). Thus, the Rad GTPase domain displays no significant changes in the overall fold compared to H-Ras and other Ras-family GTPases such as RalA, though there are several unusual features in the detailed structures which will be described below.


Figure 1
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  		Figure 1  Structure of human Rad bound to GDP. (A) Structure of the Rad GTPase domain. Molecule A is shown with {alpha}-helices (cyan) and ß-strands (magenta) with residue numbers. (B) The electron density map of the bound GDP Molecule is drawn at the 2.0 {sigma} level. The GDP model is represented as a stick model. The protein is shown as a C{alpha}-tracing tube model with side chains as thin-wire models. (C) Sequence alignment of human Rad, Gem, RalA and H-Ras. Secondary structural elements of Rad and H-Ras are indicated at the top and below, respectively. Omitted regions of the current Rad model comprise nine residues (116–124) in switch I, seven residues (147–153) in switch II, three residues (192–194) in loop A3-B5, and three (87–89) and four (255–258) residues of the N- and C-terminus, respectively, and are indicated by broken lines. Conserved G1-G5 motifs are shown below with consensus sequences and highlighted in yellow. Nonconservative residues that are important for the conformation properties and the catalytic action are found both inside the motifs (highlighted in magenta) and outside the motifs (cyan), respectively.

 

Figure 2
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  		Figure 2  Switch I flexibility. (A) Comparison of GTPase domain structures of Rad, H-Ras and RalA bound to GDP. C{alpha}-tracing representation of Rad (blue) superimposed on H-Ras (yellow) and RalA (green). (B) A closeup view of Rad helix A1 and switch I.Most of switch I (green) is disordered. Water molecules are shown as balls (red) and the GDP molecule is represented as a stick model with the magnesium ion as a large ball (gray). Hydrogen bonds are indicated by dotted lines. (C) A closeup view of H-Ras helix A1 and switch I corresponding to (B). H-Ras is the GDP-bound form (1Q21). Switch I (green) covers the GDP molecule with non-polar interactions between Phe28 and the guanine base. The switch conformation is stabilized by hydrophobic interactions centered by Ile21 and a hydrogen bond between conserved tyrosine residues Tyr32 and Tyr40.

 
The switch I region

The switch I region of small GTPases generally contains the G2 motif (FVXXYXPTXXDXY where X represents any amino acid residue) that is important for nucleotide binding. The Rad switch I region, however, lacks this motif (Fig. 1C). In our crystal, switch I was disordered and most of the residues are invisible in the current map (Fig. 2B). Helix A1, which is followed by switch I, is important for stabilization of the switch I conformation. Helix A1 of Rad is shorter than that of H-Ras, being hampered by two glycine residues (112 and 113) at the C-terminus of the helix. These small residues cause a small shift of the C-terminal part of helix A1 towards the ß-sheet (Fig. 2B). In H-Ras, Ile24 exists at the position corresponding to Gly112 of Rad and stabilizes helix A1 by making contacts with the ß-sheet (Fig. 2C). In addition to these changes in configuration, Rad helix A1 has a polar residue, Arg109, on the helix surface. This position is usually occupied by a non-polar residue, Ile21 in H-Ras, which stabilizes the loop conformation of switch I through hydrophobic interactions with two conserved residues, Val29 and Tyr32, of the G2 motif. Moreover, a hydrogen bond between conserved tyrosine residues Tyr32 and Tyr40 stabilizes the loop. The absence of the conserved valine and tyrosine residues, together with the relatively high content of mobile glycine residues, might impart flexibility to the Rad switch I region.

Rad switch I lacks the conserved phenylalanine (Phe28 of H-Ras) that traps the GDP molecule through non-polar interactions with the base and sugar. Instead, Arg109 on helix A1 serves to hold the nucleotide by forming a water-mediated hydrogen bonding network (Fig. 2B).

The switch II region

Switch II of Rad is also disordered in our crystal, suggesting conformational flexibility. The G3 motif comprises the N-terminal half of switch II. Rad possesses marked substitutions within this motif (DIWEQD) compared to the common sequence (DTAGQE) that is completely conserved in H-Ras and RalA. At the beginning of the loop, two hydrophobic residues, Ile145 and Trp146, of Rad pack against helices A2 and A3 to form a compact hydrophobic core (Fig. 3A). Ile145 interacts with Met161, which would result in unwinding of the C-terminus of helix A2. These interactions seem to induce a shift in orientation of helix A2 compared with that found in H-Ras. Rad displays a similar orientation of helix A2 to that of RalA, though the length of the helix is much shorter than that of RalA (Nicely et al. 2004). This difference in helix length is caused by the introduction of a helix breaker, Pro155, at the N-terminus of Rad helix A2.


Figure 3
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  		Figure 3  Switch II flexibility. (A) A closeup view of Rad switch II and helix A2.The loop region (147–153) of switch II (green) is disordered. Hydrogen bonds are indicated by dotted lines. (B) A closeup view of H-Ras switch II and helix A2 corresponding to (A).

 
At the middle of switch II, Rad contains two glycine residues, which would endow the loop with conformational flexibility. Switch II of H-Ras is stabilized by a salt bridge between Arg68 of the loop and Glu37 of switch I. These residues are not conserved in Rad, which might contribute to the flexibility. Rad preserves Gln148 (Gln61 of H-Ras, DTAGQE) that is essential for catalytic activity in H-Ras and other small GTPases. However, we suspect that the catalytic mechanism of GTP hydrolysis by Rad might be somewhat different from that of H-Ras. Indeed, mutation of Rad Gln148 had no effect on GTPase activity (Zhu et al. 1995). Consistent with this, the corresponding residues are asparagine and alanine in Gem and Rem, respectively. These observations suggest that the conformational properties of Rad switch II are distinct from that of H-Ras and its closely related GTPases.

The phosphate-binding loop

The phosphate-binding loop of Rad possesses a G1 motif (GXPXXGKSXL) that has a proline substitution, Pro100, at the second conserved glycine residue (G12 of Ras) of the common G1 motif (GXGXXGKSXL) (Fig. 1C). The location of this glycine residue is well known for the site of mutation to produce a transforming form of H-Ras. Crystal structures of mutated H-Ras GTPases show that the mutations cause no significant conformational change in the phosphate-binding loop. Similar results were obtained in Rad, which displays the G1 motif well overlaid on those of H-Ras and RalA with extremely small r.m.s. deviations (0.16–0.18 Å) (Fig. 4). This structural conservation is coincident with the fact that the Rad phosphate-binding loop retains main chain interactions with the ß-phosphate of GDP as in H-Ras.


Figure 4
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  		Figure 4  Comparison of the phosphate-binding loops of Rad and related small GTPases. The superimposed segments are GAPGVGKS of Rad (blue) and GAPGVGKS of G12P mutated H-Ras (magenta) and GSGGVGKS of RalA-GDP (green). The magnesium ion is represented as a large ball (orange).

 
In H-Ras, several bulky mutations of Gly12 such as G12V and G12P are known to stabilize the switch II conformation through direct contacts between the mutated bulky residue and switch II, and to define a single conformation of switch II, whereas wild-type H-Ras possesses a disordered switch II region (Krengel et al. 1990; Pai et al. 1990). A similar result was obtained for the G14V mutation in RhoA (Ihara et al. 1998). Contrary to these observations, the Rad phosphate-binding loop with the Pro100 substitution might not contribute to stabilization of the switch II conformation. Perhaps the conformational properties of Rad switch II are distinct from that of H-Ras as mentioned above. Mutation of Rad Pro100 to a glycine residue had no effect on GTPase activity (Zhu et al. 1995).

Magnesium ion binding

In our Rad-GDP complex, the magnesium ion is located at the same position as in the H-Ras-GDP complex. The superposition of Rad and H-Ras proteins yields a small deviation of 0.37 Å for magnesium ions. The ion possesses the same octahedral coordination shell that includes four water molecules, one ß-phosphate oxygen atom and the side chain of Ser105 (Ser17 of H-Ras) (Fig. 3). Conserved Ser105 is located in the G1 motif and is stabilized by conserved Asp144 (Asp57 of H-Ras) of the G3 motif. Mutation of Ser105 with asparagine in Rad abolishes GTP binding (Zhu et al. 1995). Taking into account the lack of switch I–nucleotide interactions, the magnesium ion seems to be more critical for GDP binding to Rad than in other GTPases. Moreover, switch I lacks the conserved threonine residue (Thr35 of H-Ras) that is well known to coordinate to the magnesium ion in the GTP-bound form, suggesting relatively weak binding of the magnesium ion to the GTP-bound form. This idea is consistent with the fact that Rad displays unique magnesium dependence where GDP and GTP binding were optimal at relatively high magnesium ion concentrations (1–10 mM) (Zhu et al. 1995).

The base recognition

The GDP molecule bound to Rad displays an anti-glycosyl conformation with a C2’-endo sugar pucker, basically identical to that of GDP conformations bound to other small GTPases including H-Ras. The guanine base is trapped in a hydrophobic pocket, in a manner similar to H-Ras, and participates in several interactions with conserved residues (Asn203, Lys204, Asp206 and Ala234) of the G4 and G5 motifs (Fig. 5A). The hydrogen bonding network between the base and these two motifs is the same as in H-Ras. The superposition of Rad on H-Ras yields an r.m.s. deviation of 0.95 Å for atoms of the bound GDP molecules. A notable difference in the base recognition site might be pointed out in the conformational stability of the G5 motif, in addition to the absence of contacts with the conserved phenylalanine (Phe28 of H-Ras) in switch I as mentioned above. The Rad G5 motif (ETSAAL) replaces the conserved threonine residue (EXSAXT) with a leucine residue, Leu236. In H-Ras and other small GTPases, the conserved threonine residue (Thr148 of H-Ras) plays an important role in stabilizing the base recognition loop of the G5 motif by forming intraloop hydrogen bonds (Fig. 5B). Moreover, H-Ras possesses a lysine residue at the variant position of the G5 motif (ETSAKT), while Rad possesses an alanine residue at this site. This lysine residue of H-Ras, Lys147, forms a hydrogen bond to Asp119 and contributes to stabilization of the side-chain conformation of Asp119 that participates in guanine base recognition. The absence of these interactions in Rad might impart greater flexibility in the base recognition site formed by the G4 and G5 motifs. This might be related to the observation that GTP binding to Rad was slightly inhibited in the presence of high ATP concentrations (Zhu et al. 1995).


Figure 5
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  		Figure 5  Base recognition loops. (A) A closeup view of the Rad base-recognition loops.The G4 (LVGNKSDL) and G5 (ETSAAL, green) motifs form loops that interact with the guanine base of GDP. Hydrogen bonds are indicated by thin lines. (B) A closeup view of the H-Ras base-recognition loops corresponding to (A). The G4 (LVGNKCDL) and G5 (ETSAKT, green) motifs and Phe28 from switch I form the binding site in Ras. Hydrogen bonds indicated by thick lines are absent in the Rad-GDP complex structure shown in A.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
We have for the first time determined the three-dimensional structure of a member of the RGK family GTPases. Our Rad structure revealed disordered structures of switches I and II in the GDP-bound form. We showed that the conformational flexibility of both switches is caused by non-conservative substitutions in the G2 and G3 motifs together with other substitutions in the structural elements interacting with the switches. Additionally, glycine-rich sequences of the Rad switches would contribute to the flexibility. These observations are distinct from those of H-Ras and most other small GTPases. Compared with H-Ras, there are differences in interactions between switch I and GDP. Nevertheless, the GDP molecule adopts a similar conformation to that bound to H-Ras and is located in the same position. This similarity seems to be achieved by the conserved interactions with the guanine base recognition site and the magnesium ion.

GTPase-activating proteins (GAPs) generally interact with both switches I and II of target GTPases for their stimulation activity (Vetter & Wittinghofer 2001; Hakoshima et al. 2003). Since the conformational property of Rad switches I and II is distinct from the Ras-family members including H-Ras, Rad should resist the action of Ras-specific GAPs. Indeed, known GAPs such as Ras-GAP, NF1, p190, Rap-GAP and IQGAP1 failed to stimulate Rad GTPase activity (Zhu et al. 1995). nm23-H1, found recently, was reported to exert a GAP action against Rad as well as a GEF action (Zhu et al. 1999).

Our structure of the GDP-bound form of Rad revealed uncommon structural features of the human Rad GTPase domain, especially conformational flexibility in the switch I region. These findings provide valuable clues for further biochemical and biomedical analyses of this novel small GTPase that is closely engaged in cytoskeletal rearrangement, ion channel regulation, diabetes and cancer.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Protein preparation, crystallization and data collection

Over-expression, purification and crystallization of the GTPase core domain (residues 87–258) of human Rad was carried out according to previously described methods (Yanuar et al. 2005). The crystals were grown for 2–3 days at 20 °C in a hanging drop of 1 : 1 volume mixture of 11 mg/mL protein solution and precipitant solution of 25% PEG 3350, 100 mM MOPS (pH 7.0), 25 mM MgCl2 and 10 mM DTT. The crystals were found to belong to space group P21, with unit-cell parameters a = 52.2 Å, b = 58.6 Å, c = 53.4Å, ß = 98.0Å. A diffraction data set was collected to a resolution of 1.8Å at SPring-8, Japan on beamline BL41XU using an ADSC Quantum 315 CCD detector system and the data was processed using the HKL2000 program suite (Otwinowski & Minor 1997).

Structural determination

Using the program MODELLER (Sali & Blundell 1993), starting models for molecular replacement were obtained by homology modeling with Ras-family GTPases, H-Ras (PDB code 1JAH), Rap1A (1GUA), Rap2 (3RAP) and RalA (1U90 and 1UAD). These GTPases exhibit between 34 and 37% sequence identity with Rad. The structural analysis was carried out using polyalanine models with the molecular replacement program Molrep (CCP41994). Prime-and-switch phasing was performed to remove model bias using the program RESOLVE (Terwilliger 2003) prior to creating electron denstity maps. Density modification with solvent flattening, histogram matching and-non-crystallographic symmetry averaging was also performed to improve electron density maps. Model building was performed using the program O (Jones et al. 1991). Difference Fourier maps were calculated for model building of the bound GDP molecules and picking magnesium ions. The structure was refined with the CNS program (Brünger et al. 1998). Non-crystallographic symmetry was treated during the refinement process until an R-value of 31.7% (an Rfree of 32.0%) was achieved. After several cycles of refinement, the structure was refined with an R-value of 21.4% (an Rfree of 24.0%) without any residues in disallowed regions as checked by the program PROCHECK (Laskowski et al. 1993). The results of the data collection and refinement of the structure are summarized in Table 1. The structure was inspected and rendered using the program PyMOL (DeLano & Bromberg 2004).


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  		Table 1 Crystallographic data
 


   Acknowledgements
 
We would like to thank Drs K. Kaibuchi and H. Yamaguchi for providing the human Rad cDNA and for the subcloning, respectively. This work was supported in part by a Protein 3000 project on Signal Transduction from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to T.H.). A.Y. was supported by an Iida International Student Scholarship from Japan Educational Exchanges and Services. S.S. was supported by a postdoctoral research fellowship for Young Scientists from the Japan Society for the Promotion of Science.


   Footnotes
 
Accession numbers: Atomic coordinates and structure factors have been deposited with the Protein Data Bank under accession code 2DPX.

Communicated by: Masao Tasaka

* Correspondence: E-mail: hakosima{at}bs.naist.jp


   References
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 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Beguin, P., Mahalakshmi, R.N., Nagashima, K., et al. (2006) Nuclear sequestration of beta-subunits by Rad and Rem is controlled by 14-3-3 and calmodulin and reveals a novel mechanism for Ca2+ channel regulation. J. Mol. Biol. 355, 34–46.[CrossRef][Medline]

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Krengel, U., Schlichting, L., Scherer, A., et al. (1990) Three-dimensional structures of H-ras p21 mutants: molecular basis for their inability to function as signal switch molecules. Cell 62, 539–548.[CrossRef][Medline]

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Received: 12 May 2006
Accepted: 18 May 2006




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