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Originally published In Press as doi:10.1074/jbc.M501253200 on February 23, 2005

J. Biol. Chem., Vol. 280, Issue 17, 17093-17100, April 29, 2005
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Structural Basis for the Unique Biological Function of Small GTPase RHEB*

Yadong Yu{ddagger}, Sheng Li{ddagger}, Xiang Xu{ddagger}, Yong Li§, Kunliang Guan§, Eddy Arnold¶, and Jianping Ding{ddagger}||

From the {ddagger}Key Laboratory of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences and Graduate School of the Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, China, §Life Science Institute, Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109-0606, and Center for Advanced Biotechnology and Medicine and Rutgers University Department of Chemistry and Chemical Biology, Piscataway, New Jersey 08854-5638

Received for publication, February 3, 2005 , and in revised form, February 22, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
The small GTPase Rheb displays unique biological and biochemical properties different from other small GTPases and functions as an important mediator between the tumor suppressor proteins TSC1 and TSC2 and the mammalian target of rapamycin to stimulate cell growth. We report here the three-dimensional structures of human Rheb in complexes with GDP, GTP, and GppNHp (5'-({beta},{gamma}-imide)triphosphate), which reveal novel structural features of Rheb and provide a molecular basis for its distinct properties. During GTP/GDP cycling, switch I of Rheb undergoes conformational change while switch II maintains a stable, unusually extended conformation, which is substantially different from the {alpha}-helical conformation seen in other small GTPases. The unique switch II conformation results in a displacement of Gln64 (equivalent to the catalytic Gln61 of Ras), making it incapable of participating in GTP hydrolysis and thus accounting for the low intrinsic GTPase activity of Rheb. This rearrangement also creates space to accommodate the side chain of Arg15, avoiding its steric hindrance with the catalytic residue and explaining its noninvolvement in GTP hydrolysis. Unlike Ras, the phosphate moiety of GTP in Rheb is shielded by the conserved Tyr35 of switch I, leading to the closure of the GTP-binding site, which appears to prohibit the insertion of a potential arginine finger from its GTPase-activating protein. Taking the genetic, biochemical, biological, and structural data together, we propose that Rheb forms a new group of the Ras/Rap subfamily and uses a novel GTP hydrolysis mechanism that utilizes Asn1643 of the tuberous sclerosis complex 2 GTPase-activating protein domain instead of Gln64 of Rheb as the catalytic residue.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Rheb (Ras homolog enriched in brain) is a small GTPase that was first identified in neuronal tissues and subsequently found to be ubiquitously expressed and particularly abundant in muscle and brain (1–3). The exact biological function(s) of Rheb was unknown until recently. Genetic, cell biological, and biochemical studies in both Drosophila and cultured mammalian cells have now demonstrated that Rheb functions as an important mediator between the tumor suppressor proteins TSC11 and TSC2 (tuberous sclerosis complex 1 and 2) and the mammalian target of rapamycin (mTOR) to stimulate cell growth (4–18). TSC1 and TSC2 are the protein products of the tsc1 and tsc2 genes (19, 20). Mutations in either tsc1 or tsc2 can cause an autosomal dominant disorder known as TSC that is manifested by the occurrence of different types of benign tumors in a variety of tissues and organs (21, 22). TSC1 and TSC2 form a physical and functional complex that inhibits the activation of mTOR through Rheb. mTOR phosphorylates and regulates ribosomal S6 kinase 1 and eukaryotic initiation factor 4E-binding protein 1. TSC2 contains a highly conserved GTPase-activating protein (GAP) domain at its C terminus (TSC2GAP), which is homologous to Rap1GAP (23). TSC2GAP directly down-regulates Rheb by stimulating its GTPase activity (8–10, 16, 17, 24). The mTOR-signaling pathway is a central controller of cell growth in response to growth factors, cellular energy, and nutrient levels. Rheb stimulates the phosphorylation of S6 kinase 1 and 4E-binding protein 1 through activation of mTOR signaling. Mutations in Rheb can cause cell growth inhibition, whereas overexpression of Rheb can promote an increase in cell size and alter the cell cycle. A high proportion of missense mutations of TSC2 identified in TSC patients are defective in repressing the phosphorylation of S6 kinase 1 and 4E-binding protein 1 (5, 20, 24), suggesting that the constitutive activation of mTOR/S6 kinase 1/4E-binding protein 1-signaling pathway may be one of the etiologic factors of TSC. However, the mechanism of mTOR activation by Rheb is not yet understood.

Rheb shares moderate sequence homology with other small GTPases, especially members of the Ras/Rap subfamily (about 30–40% sequence identity) and therefore has been suggested to belong to the Ras/Rap subfamily (1). However, Rheb possesses unique biological properties that differ substantially from those of other members of the Ras/Rap subfamily. For most Ras-like small GTPases, the canonical model of GAP-stimulated GTP hydrolysis primarily involves the stabilization of a conserved Gln at the catalytic site of GTPase (corresponding to Gln61 in Ras), the insertion of an arginine finger of GAP into the catalytic site, and the conformational changes and stabilization of the switch regions of the GTPase (25–27). Though Rheb contains a conserved Gln (Gln64) at the equivalent position, its mutation to Leu has limited effect on the GTPase activity of Rheb (10, 24), and the Q64L mutant Rheb functions similarly to the wild-type Rheb to inhibit the Ras transformation (28). In addition, unlike the Q61L mutant Ras, which is resistant to the deactivation of RasGAP, the Q64L Rheb is still sensitive to TSC2GAP (24). So far, both sequence comparisons and mutagenesis studies have not identified an equivalent arginine finger in TSC2GAP (24, 29). Moreover, Rheb has a very low intrinsic GTPase activity and exists in a high activation state (30). These distinct properties render Rheb a unique regulator in the mTOR-signaling pathway.

To investigate the biological function(s) of Rheb in mTOR signaling and gain insights into the potential interactions of Rheb with its upstream and downstream partners, we carried out structural studies of human Rheb (RHEB). RHEB consists of 184 amino acid residues with a molecular mass of 22 kDa (Swiss-Prot code Q15382 [GenBank] ) (2). The 169 N-terminal residues form the GTPase domain; the 15 C-terminal residues are hypervariable with a flexible structure and comprise a conserved carboxyl CAAX motif that plays important roles in the farnesylation of Rheb and its association with membrane (8, 10). We report here the crystal structures of the GTPase domain of RHEB in complexes with GDP, GTP, and a GTP analog, GppNHp, at 2.0, 2.8, and 2.65 Å resolution, respectively. Structural comparison of Rheb with other small GTPases reveals novel structural features of Rheb and provides a molecular basis for its unique biological properties. This knowledge is valuable in understanding the biological function of Rheb in the mTOR-signaling pathway and may provide opportunities for the design of novel drugs for treating TSC and other related tumors.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Protein Expression and Purification and Crystallization—Similar to the structural studies of other small GTPases, we constructed a truncated form of RHEB (residues 1–169) (referred to as RHEB hereafter) that omitted the 15 C-terminal hypervariable residues to facilitate crystallization. Expression and purification of RHEB and the crystallization and diffraction data collection of RHEB in complexes with GDP, GTP, and GppNHp are described elsewhere (31). Statistics of the diffraction data used for structure determination and refinement of the RHEB complexes are summarized in Table I.


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  		TABLE I
Statistics of diffraction data and refinement

 
Structure Determination and Refinement—The structure of the RHEB-GDP complex was solved using the molecular replacement method as implemented in the program CNS (32), with the structures of two most homologous small GTPases, human Rap2A and -1A (Protein Database accession numbers 1KAO [PDB] and 1C1Y, respectively) (33, 34), as the search models. The structures of the RHEB-GppNHp and RHEB-GTP complexes were solved using the molecular replacement method with the refined structure of the RHEB-GDP complex as the search model. Structure refinement was carried out using the program CNS, with 5% of the reflections omitted for free R factor calculation. In the initial difference Fourier maps, there were strong residual electron density peaks at the nucleotide-binding site that matched substrate GDP (or GppNHp or GTP) and an Mg2+ ion very well. Conformational changes were observed in both switch I and II regions and in several surface-exposed loops in different RHEB complexes. Those regions were remodeled based on the electron density. Electron density peaks in difference Fourier maps at a height of >3{sigma} were assigned as water molecules in later refinement stages if they had reasonable geometry in relation to hydrogen bond donors or acceptors. Model building was performed with the program O (35) and guided by SIGMAA-weighted 2Fo – Fc and Fo – Fc maps and composite omit maps.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Overall Structure of RHEB—Crystal structures of the GTPase domain (residues 1–169) of RHEB were determined in complexes with GDP, GTP, and GppNHp at 2.0, 2.8, and 2.65 Å resolution, respectively. A summary of the structure refinement and the final model statistics is given in Table I. Fig. 1 shows representative SIGMAA-weighted 2Fo – Fc composite omit maps at the switch II region in different complexes. The overall structure of RHEB resembles that of other small GTPases (Fig. 2). Structures of the three different RHEB complexes are very similar to each other. Superposition of the RHEB-GDP complex onto the RHEB-GppNHp and RHEB-GTP complexes yielded an r.m.s. deviation value of 0.76 and 0.79 Å, respectively, and superposition of the RHEB-GppNHp complex onto the RHEB-GTP complex gave an r.m.s. deviation value of 0.31 Å.



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  		FIG. 1.
Representative SIGMAA-weighted 2Fo – Fc composite omit maps (1 {sigma} contour level) at the switch II region. a, the RHEB-GDP complex. b, the RHEB-GTP complex. c, the RHEB-GppNHp complex. The final coordinates of the structures are shown as ball-and-stick models.

 



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  		FIG. 2.
Structure of RHEB and its comparison with other small GTPases. a, overall structure of the RHEB-GppNHp complex. The secondary structure elements of RHEB are named after those of human Ras, except that helix {alpha}2 of Ras is replaced by a short 310 helix ({eta}1). Switches I and II are colored in green, and the P loop is in blue. The bound GppNHp is shown as a ball-and-stick model, Mg2+ ion as a cyan sphere, and the two conserved water molecules as red spheres. b, comparison of RHEB with Ras showing the conformational differences in the switch I and II regions. The RHEB-GppNHp complex is shown in green, the RHEB-GDP complex in magenta, the Ras-GppNHp complex in red, and the Ras-GDP complex in gold. The P loop is colored in blue. For clarity, only the GppNHp substrate and the Mg2+ ion in the RHEB-GppNHp complex are shown. c, structure-based sequence alignment of RHEB with six other small GTPases from human with high sequence homology. The Swiss Protein Database codes of the structures used in comparison are listed in Table II. Invariant residues are highlighted in shaded red boxes and conserved are in open red boxes. The secondary structures of RHEB and Ras are above the alignments.

 
Rheb shares moderate sequence homology with Ras and Rap of the Ras/Rap subfamily and with several other small GTPases outside the Ras/Rap subfamily, thus rendering the classification of Rheb less conclusive (Table II and Fig. 2C). Structural comparisons between RHEB and those small GT-Pases show that RHEB is more closely related to Ras and Rap than to RhoA and Rab5A (Table II). Superposition of RHEB with those small GTPases yielded an r.m.s. deviation value in the range of 1–1.6 Å. The major structural differences among different RHEB complexes and between RHEB and the other small GTPases occur mainly in the switch I and switch II regions that are involved in recognition and interaction with GAPs, guanine nucleotide exchange factors, and effectors (27, 36). Based on sequence alignment, the switch regions of RHEB are defined as residues 33–41 (switch I), corresponding to residues 30–38 of Ras, and residues 63–79 (switch II), corresponding to residues 60–76 of Ras.


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  		TABLE II
Sequence and structural comparison of RHEB with six other small GTPases

Small GTPases from human that share high sequence homology with RHEB are identified using BLAST search with NCBI data base. Structures of those small GTPases in their complexes with GDP, GTP, or its analogs GppNHp or GTP{gamma}S (guanosine 5'-O-(thiotriphosphate)) are retrieved from the Protein Data Bank (accession codes are listed in parentheses). No structure of human Rab21 in any state is available yet.

 
In all three RHEB complexes, the electron density maps unambiguously showed the presence of a nucleotide (GDP, GTP, or GppNHp) and an Mg2+ ion, which is required for the GTPase activity, at the catalytic active site. The bound substrate has extensive hydrophobic and hydrophilic interactions with the surrounding residues. Most of these interactions have also been observed in other small GTPases in their complexes with GTP, GTP analogs, or GDP. In the RHEB-GppNHp structure, the Mg2+ ion is coordinated by six oxygens in a slightly distorted octahedral geometry: one each from the {beta}- and {gamma}-phosphates of the substrate (2.6 and 2.4 Å, respectively), Ser20 O-{gamma} (2.3 Å), Thr38 O-{gamma} (2.6 Å), and two water molecules (Wat1 and Wat2, 2.2 and 2.5 Å, respectively) (Figs. 2 and 3). A conserved water molecule (Wat3) that is proposed to initiate the nucleophilic attack on the {gamma}-phosphate of the nucleotide in GTP hydrolysis was stabilized by hydrophilic interactions with the main chain atoms of Gly63 (N, 2.9 Å and O, 4.1 Å), Thr38 (N, 3.9 Å and O, 3.8 Å), and the {gamma}-phosphate of GppNHp (2.6, 3.1, and 2.9 Å with O1G, O2G, and O3G, respectively). The {gamma}-phosphate of GppNHp had hydrogen-bonding interactions with several conserved residues nearby, including Lys19 (N-{zeta} atom, 2.9 Å), Tyr35 (O-{eta} atom, 2.6 Å), Thr38 (N atom, 3.2 Å), and Gly63 (N atom, 3.2 Å). The phosphates of the substrate were partially shielded by switch I, primarily by the aromatic side chain of Tyr35. No significant change was observed in the position and conformation of the nucleotide and the conformation of the protein between the RHEB-GTP complex and the RHEB-GppNHp complex.



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  		FIG. 3.
Structure of the catalytic active site region. a, comparison of RHEB in complexes with GppNHp and GDP. b, comparison of RHEB with Ras in their complexes with GppNHp. The color-coding scheme is the same as in Fig. 2. Residues in the RHEB-GppNHp complex are shown with yellow side chains and labels; residues in other complexes are shown with gray side chains without labels. c, molecular surface of the catalytic active site in the structure of the RHEB-GppNHp complex (left panel) and in the structure of the Ras-GppNHp complex (right panel).

 
In the RHEB-GDP structure, the Mg2+ ion shifted by 0.9 Å toward the substrate and was coordinated by the O-{gamma} atom of Ser20, the O3B atom of the GDP {beta}-phosphate, and four water molecules in a distorted octahedral geometry. One water molecule occupied the position taken by one {gamma}-phosphate oxygen of GTP and another occupied the position taken by the Thr38 O-{gamma} atom. Because of the flipped-down conformation of switch I (see discussion later), the bound GDP was largely exposed to solvent, and several water molecules occupied the space taken by switch I in the GppNHp-bound complex and made hydrogen bonds with the phosphate and ribose moieties of GDP.

Arg15 of Rheb Is Not Directly Involved in GTP Hydrolysis— Comparison of RHEB with other small GTPases indicated that the phosphate-binding loop (P loop) has a very stable structure among different small GTPases and between their active and inactive forms (the r.m.s. deviation of C-{alpha} atoms between them is in the range of 0.17 to 0.30 Å) (Figs. 2 and 3). Members of the Ras/Rap subfamily contain a highly conserved Gly in the P loop corresponding to Gly12 in Ras. Mutation of Gly12 to any other residue except Pro impairs the intrinsic GTPase activity of Ras, making the mutant Ras constitutively active (26, 37, 38). In particular, the G12R mutant Ras confers drastically reduced intrinsic GTPase activity and is resistant to inactivation by RasGAP. This mutation interferes with GTP hydrolysis by steric hindrance between the bulky side chain of Arg12 and the side chain of Gln61, pushing Gln61 into a catalytically incompetent conformation (38). Interestingly, Rheb contains a conserved Arg15 at the equivalent position, and the wild-type Rheb has very low intrinsic GTPase activity and a high basal GTP level (~50%, which is more than 10-fold higher than that of Ras), leading to the suggestion that Arg15 of Rheb is responsible for those properties (30). However, the wild-type Rheb can still be inactivated by TSC2GAP (8–10, 16, 17), and mutation of Arg15 to Gly, Val, or Pro does not affect its activation level in the absence of TSC2GAP (24, 30).

In the GTP- and GppNHp-bound RHEB structures, the side chain of Arg15 was oriented toward solvent and stacked on top of the aromatic side chain of Tyr14 (Fig. 3). Its side chain N-{eta}2 atom formed a salt bridge with the side chain O-{delta}2 atom of Asp65 (2.8 Å), and its N-{eta}1 atom had a hydrophilic interaction with the main chain O atom of Gly63 (3.8 Å). Because of the unfolding of switch II and the displacement of Gln64 (see discussion later), the side chain of Arg15 did not have steric hindrance with the side chain of Gln64, as seen between Arg12 and Gln61 in mutated Ras or interaction with the nucleotide and Mg2+. In the RHEB-GDP complex, the Arg15 side chain was oriented slightly away from switch II, and its interactions with Asp65 and Gly63 were disrupted. Instead, the N-{eta}2 atom of Arg15 formed a hydrogen bond with the O-{eta} atom of Tyr14 (3.1 Å). In both cases, the side chain of Arg15 was not involved in interactions with either the nucleotide or with Gln64, suggesting that Arg15 is not directly involved in GTP hydrolysis and, therefore, is not responsible for its high basal GTP level. Instead, it might have a different functional role in GTP hydrolysis. Recent biochemical data have shown that the R15G mutant Rheb is partially resistant to inactivation by TSC1/TSC2, suggesting that Arg15 plays a role in the interaction of Rheb with TSC2GAP (24).

Switch I Undergoes Major Conformational Change during GTP/GDP Cycling—Switch I of small GTPases played important roles in recognizing and interacting with their effectors, and the differences in primary sequences and structures of switch I determined the specificity of small GTPases to their effectors. Similar to other small GTPases, switch I of RHEB had relatively high flexibility with a high average B factor (50.0 versus 31.3 Å2 for the whole protein in the GppNHp-bound complex, 46.5 versus 38.5 Å2 in the GTP-bound complex, and 55.0 versus 28.1 Å2 in the GDP-bound complex, respectively). Sequence comparison indicated that switch I of RHEB is more similar to those of Ras and Rap than to those of Rab5A and RhoA (Fig. 2C). Six of the nine residues in switch I were identical between Rheb and Ras/Rap, and two of the three non-identical residues were highly conserved among different Rhebs. On the other hand, only three of the nine residues were identical between Rheb and Rho, and only one was identical between Rheb and Rab. Structural comparison also showed that switch I of RHEB is very similar to those of Ras and Rap but has marked differences from those of RhoA and Rab5A in their respective complexes with GDP, GTP, or GppNHp.

Like other small GTPases, switch I in the GDP-bound RHEB had profound conformational change compared with the GTP- or GppNHp-bound RHEB, although the position of the nucleotide had no evident difference (Fig. 2). In particular, Tyr35 and Ile39 flipped up toward the inside of the protein, whereas Thr38 flipped down toward the outside (Fig. 3). Similar conformational change was observed in Ras and Rap between the GDP-bound form and the GTP-bound form. However, the conformational differences of switch I between RHEB and Ras/Rap were more remarkable in their complexes with GDP than in their complexes with GTP or GppNHp, especially at the N-terminal part of switch I (Fig. 2). Residues 32–36 of RHEB were positioned further outward from GDP; the C-{alpha} atoms of these residues differed by 1.4, 2.4, 2.8, 1.0, and 0.8 Å, respectively, relative to those in the Ras-GDP complex (or by comparable distances in the Rap2A-GDP complex). The charged side chains of Glu31 in Ras and Lys31 in Rap have been proposed to determine the specificity for their effectors (39). The equivalent position was occupied by a conserved, short, and non-charged residue Ser34 in Rhebs. These differences might suggest that Rheb has a different effector from that of Ras/Rap and/or interacts with its effector in a different way.

Switch II Adopts a Unique Conformation and Maintains a Relatively Stable Structure—The major difference between RHEB and other small GTPases occurred in the switch II region (Figs. 2 and 3). In most small GTPases, switch II assumes a long {alpha}-helical conformation (corresponding to {alpha}2 in Ras) in their complexes with GTP or GppNHp but differs slightly in their complexes with GDP with the N-terminal part disordered to varying extents (36). During the GTP/GDP cycling, switch II underwent marked conformational change. Surprisingly, in the structures of RHEB complexes with GTP and GppNHp, switch II assumed an unraveled conformation instead of the {alpha}-helical conformation, and the entire region had well defined electron density with a relatively low average B factor (35.0 Å2 versus 31.3 Å2 for the whole protein in the GppNHp-bound complex and comparable values in the GTP-bound complex) (Figs. 2 and 3). Only residues 72–74 formed a short 310 helix ({eta}1), whereas residues 63–71 adopted an extended loop conformation. The N-terminal loop was positioned near the P loop (residues 13–15) and the N-terminal part of helix {alpha}3 and made extensive hydrophobic and hydrophilic interactions with these structural elements. The C-terminal portion was parallel to helix {alpha}3 and had largely hydrophobic interactions with the C-terminal part of the helix. These interactions appeared to stabilize the unique switch II conformation of Rheb. Glu66 at the tip of the loop was positioned >8 Å (C-{alpha} position) away from the equivalent residue (Glu63) in Ras or Rap (Fig. 3). Gln64 was displaced by more than 4 Å (C-{alpha} position) compared with the corresponding residue Gln61 in Ras or Thr61 in Rap. The polar side chain of Gln64 was pointed away from the nucleotide-binding site and buried in a hydrophobic pocket formed by Leu12, Phe70, Pro71, Tyr74, and Ile99 and, hence, formed no interaction with GTP (or GppNHp), the nucleophilic water molecule (Wat3), or other residues at the catalytic active site.

More surprisingly, switch II maintained a similar conformation with fairly ordered electron density and a relatively low average B factor in the RHEB-GDP complex (38.2 versus 28.1 Å2 for the whole protein) and underwent minor conformational changes during the transition of GTP/GDP (Figs. 2 and 3). One marked difference between the GTP- and GDP-bound forms was a flip of the main chain carbonyl group of Ala62, resulting in changes of the main chain torsion angles of the Ala62-Gly63 dipeptide and a displacement of Gly63 (1.5 Å in C-{alpha} position). The side chain of Gln64 was slightly reoriented and made hydrogen-bonding interactions with the main chain amide group of Gly13 and the side chains of Thr61 and Tyr74. Meanwhile, the side chain of Arg15 was also slightly reoriented, and its hydrophilic interactions with the side chain of Asp65 and the main chain carbonyl of Gly63 were disrupted. Another notable difference was the conformational change of the C-terminal part of switch II; the 310 helix in the GDP-bound complex was displaced by about 2 Å compared with the GTP- or GppNHp-bound complex, and Tyr74 moved closer to Phe70, making the hydrophobic interactions with helix {alpha}3 much tighter.

The three different RHEB complexes with GTP, GppNHp, and GDP crystallized with different space group symmetries and at different crystallization conditions (31). Analysis of crystal packing indicated that only a few residues of switch II were involved in weak intermolecular contacts. Therefore, it is unlikely that the unique conformation of switch II is caused by crystal packing; instead, it should be an inherited feature of RHEB and related to its biological function.

Unique Switch II Conformation Is Responsible for the Low Intrinsic GTPase Activity of Rheb—Both biochemical and cell biological data have shown that Rheb has a very low intrinsic GTPase activity and a high basal GTP level, leading to the suggestion that the wild-type Rheb exists as a constitutively active protein (28, 30). Our structural data indicated that Arg15 was not directly involved in GTP hydrolysis and was not responsible for the low GTPase activity of Rheb (see discussion above). What is the molecular basis of the low GTPase activity of Rheb? Most small GTPases contain a conserved Gln residue in switch II, corresponding to Gln61 of Ras and Gln64 of RHEB, respectively. Gln61 of Ras is directly involved in GTP hydrolysis by polarizing a conserved water molecule that carries out nucleophilic attack on the {gamma}-phosphate of GTP (26, 40). Mutation of Gln61 in Ras caused a drastic increase of the basal GTP level and conferred resistance to RasGAP inactivation, generating a constitutively active Ras mutant. Because of the distinct switch II conformation in the RHEB structures, the main chain of Gln64 was displaced by more than 4 Å from its counterpart in Ras, and its polar side chain was buried inside a hydrophobic core and made no interactions with the nucleotide or the functionally important residues at the catalytic active site. Thus, Gln64 of RHEB is unlikely to participate in GTP hydrolysis, which is consistent with the biochemical data showing that the Q64L mutation does not have a significant effect on the GTPase activity of Rheb (10, 24). In addition, the Q64L mutant Rheb is still sensitive to TSC2GAP (24). Taken together, these data lead us to propose that the unique switch II conformation and the inability of Gln64 to participate in GTP hydrolysis are directly responsible for the low GTPase activity of Rheb and account for its high activation state.

A Closed GTP-binding Site May Prohibit Insertion of a Potential Arginine Finger—In contrast to RasGAP, no equivalent Arg residue has been identified in TSC2GAP or Rap1GAP (24, 29). Although several conserved arginines of TSC2GAP are shown to be essential for its GAP activity on Rheb, they are not equivalent to the arginine finger of RasGAP (24). Why do Rheb and Rap not need an arginine finger from their GAPs? The crystal structures of RHEB provide a potential answer for this question. Although the conformation of switch I in RHEB was very similar to that of other small GTPases in their complexes with GTP or GppNHp, there was a notable difference in the conformation of a conserved Tyr in this region, corresponding to Tyr35 of RHEB, Tyr32 of Rap, and Tyr32 of Ras. In the structure of the Ras-GppNHp complex, the side chain of Tyr32 was oriented away from the nucleotide and made no interaction with GppNHp, and there is an entrance to access the phosphate groups of the nucleotide (40) (Fig. 3C). In the structure of the Ras-RasGAP complex, Tyr32 retained a similar conformation, and the arginine finger of RasGAP inserts into the catalytic site of Ras through the entrance and forms hydrogen bonds with the phosphate groups of GTP (26). However, in the structures of Rheb in complexes with GTP or GppNHp, Tyr35 was positioned ~1.5 Å (C-{alpha} position) closer to the nucleotide; its side chain moved by >4 Å to cover the top of the phosphate groups of GTP compared with Ras Tyr32, and its O-{eta} atom made a hydrogen bond with the {gamma}-phosphate of GTP (or GppNHp) (Fig. 3C). In the GTP- or GppNHp-bound Rap structures, Tyr32 of Rap had a similar conformation as Tyr35 of Rheb. The covering of Tyr35 of Rheb or Tyr32 of Rap on the phosphate moiety of GTP leads to the closure of the GTP-binding site, which may not only prevent the nucleotide from dissociation but also block the access of a potential arginine finger to the phosphate groups of the nucleotide. This closed conformation of the GTP-binding site may be an inherited feature of Rheb and Rap and possibly the molecular basis of why no arginine finger is required from their GAPs. Nevertheless, it cannot be excluded that an unidentified residue of TSC2GAP might play a similar role as that of the arginine finger of RasGAP to neutralize the negative charge on the phosphate groups of GTP. The switch I region of small GTPases usually has high flexibility, and the conserved Tyr residue was thereby disordered to some extent. Thus, it is possible that the closed GTP-binding site in Rheb might open transiently to allow a potential arginine finger to insert into the catalytic active site.

A Possible GTP Hydrolysis Mechanism—In the canonical model of GAP-stimulated GTP hydrolysis mechanism, GAP accelerates the GTP hydrolysis of small GTPase by providing an arginine finger (25, 26). The side chain of Arg forms hydrogen bonds with the {beta}-{gamma} bridging oxygen of GTP to stabilize the transition state, and its main chain amide group forms a hydrogen bond with the side chain of the catalytic Gln61 of Ras to help the latter align with the hydrophilic water. The interaction of GAP with Ras also forces conformational changes that stabilized the switch regions and promote a transition toward the intermediate state of the hydrolysis reaction. Gln64 of Rheb appears to be non-catalytic and TSC2GAP appears to contain no conserved arginine residue equivalent to the arginine finger of RasGAP. The structures of Rheb provide a possible molecular basis for why Gln64 of Rheb is not involved in GTP hydrolysis and why no arginine finger is required from TSC2GAP. How does TSC2GAP stimulate GTP hydrolysis of Rheb? Biochemical and structural data have shown that RapGAP uses a catalytic asparagine (Asn290) to stimulate GTP hydrolysis of Rap (29). TSC2GAP has an equivalent residue (Asn1643), and mutation of this residue eliminates its GAP activity on Rheb (9, 17, 24). It is possibile that Asn1643 of TSC2GAP may act as the catalytic residue in stimulating the GTP hydrolysis of Rheb in a similar manner as Asn290 of RapGAP. However, like other small GTPases and their GAPs, interaction of TSC2GAP with RHEB may induce conformational changes of both proteins and rigidify the P loop and the switch regions of Rheb to facilitate the GTP hydrolysis reaction. These conformational changes might be able to bring Gln64 into the catalysis. Therefore, we cannot unequivocally exclude a potential functional role of Gln64 of Rheb and an alternative mechanism of GTP hydrolysis in the presence of TSC2GAP. A crystal structure of Rheb in complex with TSC2GAP will reveal detailed information about the interaction between them and the potential conformational changes of both proteins and provide insight into the catalytic mechanism of TSC2GAP-stimulated GTP hydrolysis of Rheb.

Rheb shares moderate sequence homology with Ras and Rap of the Ras/Rap subfamily and with several other small GTPases, including Rab21 and Rab5A of the Rab subfamily and RhoA of the Rho subfamily. Our structural data indicate that Rheb is more closely related to Ras and Rap than to Rab5A and RhoA and belongs to the Ras/Rap subfamily, which is consistent with the phylogenetic analysis results (1, 41). Moreover, Rheb possesses distinct biological and biochemical properties and unique structural features, especially in the functionally important regions that are substantially different from other members of the Ras/Rap subfamily. Taking the genetic, biochemical, biological, and structural data together, we propose that Rheb forms a new group of the Ras/Rap subfamily and might use a novel GTP hydrolysis mechanism that utilizes Asn1643 of TSC2GAP instead of Gln64 of Rheb as the catalytic residue in GTP hydrolysis.


   FOOTNOTES
 
* This work was supported by National Natural Science Foundation of China Grants 30125011, 30170223, and 30130080, Ministry of Science and Technology of China Grants 2002BA711A13, 2004AA235091, 2004CB720102, and 2004CB520801, and Chinese Academy of Sciences Grant KSCX1-SW-17. 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 atomic coordinates and structure factors (codes 1XTQ, 1XTR, and 1XTS) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/). Back

|| To whom correspondence should be addressed. Tel.: 86-21-54921619; Fax: 86-21-54921116; E-mail: jpding{at}sibs.ac.cn.

1 The abbreviations used are: TSC, tuberous sclerosis complex; GAP, GTPase-activating protein; TSC2GAP, GAP domain of TSC2; mTOR, mammalian target of rapamycin; GppNHp, 5'-({beta},{gamma}-imide)triphosphate; r.m.s., root mean square. Back



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
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