FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2150953043> ?p ?o ?g. }

• W2150953043 endingPage "1539" @default.
• W2150953043 startingPage "1528" @default.
• W2150953043 abstract "Rheb, an activator of mammalian target of rapamycin (mTOR), displays low intrinsic GTPase activity favoring the biologically activated, GTP-bound state. We identified a Rheb mutation (Y35A) that increases its intrinsic nucleotide hydrolysis activity ∼10-fold, and solved structures of both its active and inactive forms, revealing an unexpected mechanism of GTP hydrolysis involving Asp65 in switch II and Thr38 in switch I. In the wild-type protein this noncanonical mechanism is markedly inhibited by Tyr35, which constrains the active site conformation, restricting the access of the catalytic Asp65 to the nucleotide-binding pocket. Rheb Y35A mimics the enthalpic and entropic changes associated with GTP hydrolysis elicited by the GTPase-activating protein (GAP) TSC2, and is insensitive to further TSC2 stimulation. Overexpression of Rheb Y35A impaired the regulation of mTORC1 signaling by growth factor availability. We demonstrate that the opposing functions of Tyr35 in the intrinsic and GAP-stimulated GTP catalysis are critical for optimal mTORC1 regulation. Rheb, an activator of mammalian target of rapamycin (mTOR), displays low intrinsic GTPase activity favoring the biologically activated, GTP-bound state. We identified a Rheb mutation (Y35A) that increases its intrinsic nucleotide hydrolysis activity ∼10-fold, and solved structures of both its active and inactive forms, revealing an unexpected mechanism of GTP hydrolysis involving Asp65 in switch II and Thr38 in switch I. In the wild-type protein this noncanonical mechanism is markedly inhibited by Tyr35, which constrains the active site conformation, restricting the access of the catalytic Asp65 to the nucleotide-binding pocket. Rheb Y35A mimics the enthalpic and entropic changes associated with GTP hydrolysis elicited by the GTPase-activating protein (GAP) TSC2, and is insensitive to further TSC2 stimulation. Overexpression of Rheb Y35A impaired the regulation of mTORC1 signaling by growth factor availability. We demonstrate that the opposing functions of Tyr35 in the intrinsic and GAP-stimulated GTP catalysis are critical for optimal mTORC1 regulation. Tyr35 autoinhibits intrinsic GTPase activity of Rheb and maintains activation Rheb Y35A efficiently hydrolyzes GTP using Asp65 rather than Gln64 Tyr35 and Asp65 are indispensable for sensitivity of Rheb to TSC2GAP function Regulation of mTORC1 by growth factors depends on Rheb Tyr35 and Asp65 Small GTPases act as molecular switches to regulate diverse cellular functions. When bound to guanosine triphosphate (GTP), they adopt an “on” conformation that elicits a biological response. GTP hydrolysis is accompanied by a conformational change into a GDP-bound “off” conformation. Cycling between the active and inactive states of each GTPase is a result of the intrinsic nucleotide hydrolysis and exchange rates, and regulatory proteins that catalyze these processes. GTPase-activating proteins (GAPs) stimulate GTP hydrolysis, whereas guanine nucleotide exchange factors (GEFs) mediate the displacement of GDP, allowing a new GTP molecule to bind (Bos et al., 2007Bos J.L. Rehmann H. Wittinghofer A. GEFs and GAPs: critical elements in the control of small G proteins.Cell. 2007; 129: 865-877Abstract Full Text Full Text PDF PubMed Scopus (1303) Google Scholar). GTPase proteins possess either complete or partial catalytic machinery for hydrolysis of GTP. In most cases an electronegative group is used for stabilization/polarization of the hydrolytic water for in-line nucleophilic attack of the γ-phosphate (Li and Zhang, 2004Li G. Zhang X.C. GTP hydrolysis mechanism of Ras-like GTPases.J. Mol. Biol. 2004; 340: 921-932Crossref PubMed Scopus (70) Google Scholar; Maegley et al., 1996Maegley K.A. Admiraal S.J. Herschlag D. Ras-catalyzed hydrolysis of GTP: a new perspective from model studies.Proc. Natl. Acad. Sci. USA. 1996; 93: 8160-8166Crossref PubMed Scopus (197) Google Scholar). In most Ras and Rho subfamily GTPases, this is achieved by the carboxamide oxygen of a conserved Gln in a dynamic loop called switch II. Ras and Rho GAPs work by stabilizing this Gln in a catalytic conformation, whereas an Arg residue referred to as an “Arginine finger” neutralizes the developing negative charge on the α- and β-phosphates of GTP (Scheffzek et al., 1997Scheffzek K. Ahmadian M.R. Kabsch W. Wiesmüller L. Lautwein A. Schmitz F. Wittinghofer A. The Ras-RasGAP complex: structural basis for GTPase activation and its loss in oncogenic Ras mutants.Science. 1997; 277: 333-338Crossref PubMed Scopus (1195) Google Scholar). In other systems, such as Rap-RapGAP, a catalytic asparagine is provided in trans by the GAP (Scrima et al., 2008Scrima A. Thomas C. Deaconescu D. Wittinghofer A. The Rap-RapGAP complex: GTP hydrolysis without catalytic glutamine and arginine residues.EMBO J. 2008; 27: 1145-1153Crossref PubMed Scopus (91) Google Scholar). Ras homolog enriched in brain (Rheb) is a key regulator of the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) signaling pathway (Dunlop et al., 2009Dunlop E.A. Dodd K.M. Seymour L.A. Tee A.R. Mammalian target of rapamycin complex 1-mediated phosphorylation of eukaryotic initiation factor 4E-binding protein 1 requires multiple protein-protein interactions for substrate recognition.Cell. Signal. 2009; 21: 1073-1084Crossref PubMed Scopus (65) Google Scholar; Inoki et al., 2003Inoki K. Li Y. Xu T. Guan K.L. Rheb GTPase is a direct target of TSC2 GAP activity and regulates mTOR signaling.Genes Dev. 2003; 17: 1829-1834Crossref PubMed Scopus (1418) Google Scholar). Rheb-GTP promotes phosphorylation of mTORC1 targets, resulting in enhanced protein translation and cellular growth (Garami et al., 2003Garami A. Zwartkruis F.J. Nobukuni T. Joaquin M. Roccio M. Stocker H. Kozma S.C. Hafen E. Bos J.L. Thomas G. Insulin activation of Rheb, a mediator of mTOR/S6K/4E-BP signaling, is inhibited by TSC1 and 2.Mol. Cell. 2003; 11: 1457-1466Abstract Full Text Full Text PDF PubMed Scopus (848) Google Scholar). Rheb has an unusually slow intrinsic GTPase activity, which is regulated by the GAP activity of tuberous sclerosis complex 2 (TSC2), a tumor suppressor frequently inactivated in human patients with the tumor predisposition syndrome tuberous sclerosis (Garami et al., 2003Garami A. Zwartkruis F.J. Nobukuni T. Joaquin M. Roccio M. Stocker H. Kozma S.C. Hafen E. Bos J.L. Thomas G. Insulin activation of Rheb, a mediator of mTOR/S6K/4E-BP signaling, is inhibited by TSC1 and 2.Mol. Cell. 2003; 11: 1457-1466Abstract Full Text Full Text PDF PubMed Scopus (848) Google Scholar; Tee et al., 2003Tee A.R. Manning B.D. Roux P.P. Cantley L.C. Blenis J. Tuberous sclerosis complex gene products, Tuberin and Hamartin, control mTOR signaling by acting as a GTPase-activating protein complex toward Rheb.Curr. Biol. 2003; 13: 1259-1268Abstract Full Text Full Text PDF PubMed Scopus (944) Google Scholar). Rheb overexpression has been observed in certain cancer cell lines (Eom et al., 2008Eom M. Han A. Yi S.Y. Shin J.J. Cui Y. Park K.H. RHEB expression in fibroadenomas of the breast.Pathol. Int. 2008; 58: 226-232Crossref PubMed Scopus (25) Google Scholar; Im et al., 2002Im E. von Lintig F.C. Chen J. Zhuang S. Qui W. Chowdhury S. Worley P.F. Boss G.R. Pilz R.B. Rheb is in a high activation state and inhibits B-Raf kinase in mammalian cells.Oncogene. 2002; 21: 6356-6365Crossref PubMed Scopus (116) Google Scholar; Nardella et al., 2008Nardella C. Chen Z. Salmena L. Carracedo A. Alimonti A. Egia A. Carver B. Gerald W. Cordon-Cardo C. Pandolfi P.P. Aberrant Rheb-mediated mTORC1 activation and Pten haploinsufficiency are cooperative oncogenic events.Genes Dev. 2008; 22: 2172-2177Crossref PubMed Scopus (98) Google Scholar), and constitutively activated Rheb mutants can induce oncogenic transformation in cell culture (Jiang and Vogt, 2008Jiang H. Vogt P.K. Constitutively active Rheb induces oncogenic transformation.Oncogene. 2008; 27: 5729-5740Crossref PubMed Scopus (53) Google Scholar). The low intrinsic GTPase activity of Rheb has been attributed to the catalytically incompetent conformation of Gln64 (Yu et al., 2005Yu Y. Li S. Xu X. Li Y. Guan K. Arnold E. Ding J. Structural basis for the unique biological function of small GTPase RHEB.J. Biol. Chem. 2005; 280: 17093-17100Crossref PubMed Scopus (62) Google Scholar), which is homologous to Ras Gln61, but does not participate in GTP hydrolysis (Li et al., 2004Li Y. Inoki K. Guan K.L. Biochemical and functional characterizations of small GTPase Rheb and TSC2 GAP activity.Mol. Cell. Biol. 2004; 24: 7965-7975Crossref PubMed Scopus (188) Google Scholar; Marshall et al., 2009Marshall C.B. Ho J. Buerger C. Plevin M.J. Li G.Y. Li Z. Ikura M. Stambolic V. Characterization of the intrinsic and TSC2-GAP-regulated GTPase activity of Rheb by real-time NMR.Sci. Signal. 2009; 2: ra3Crossref PubMed Scopus (48) Google Scholar). TSC2GAP is thought to utilize Asn1643 to promote GTP hydrolysis by substituting for Gln64 in an “Asn thumb”-type mechanism (Inoki et al., 2003Inoki K. Li Y. Xu T. Guan K.L. Rheb GTPase is a direct target of TSC2 GAP activity and regulates mTOR signaling.Genes Dev. 2003; 17: 1829-1834Crossref PubMed Scopus (1418) Google Scholar; Marshall et al., 2009Marshall C.B. Ho J. Buerger C. Plevin M.J. Li G.Y. Li Z. Ikura M. Stambolic V. Characterization of the intrinsic and TSC2-GAP-regulated GTPase activity of Rheb by real-time NMR.Sci. Signal. 2009; 2: ra3Crossref PubMed Scopus (48) Google Scholar) similar to that of RapGAP (Scrima et al., 2008Scrima A. Thomas C. Deaconescu D. Wittinghofer A. The Rap-RapGAP complex: GTP hydrolysis without catalytic glutamine and arginine residues.EMBO J. 2008; 27: 1145-1153Crossref PubMed Scopus (91) Google Scholar). Here, utilizing site-directed mutagenesis, crystallography, and real-time NMR-based GTPase assays, we discovered that Rheb Tyr35, a residue that is highly conserved across the small GTPase superfamily (Wennerberg et al., 2005Wennerberg K. Rossman K.L. Der C.J. The Ras superfamily at a glance.J. Cell Sci. 2005; 118: 843-846Crossref PubMed Scopus (1009) Google Scholar), maintains the high activation state of Rheb by inhibiting intrinsic GTP hydrolysis. Mutation of this residue substantially accelerated intrinsic nucleotide hydrolysis through a catalytic mechanism that did not require Gln64 but also conferred resistance to the activity of TSC2. Crystal structures of Rheb Y35A led us to identify the backbone carbonyl of Thr38 and side chain of Asp65 as candidate residues that contribute to the intrinsic GTPase activity. Mutagenesis studies confirm that Asp65 contributes significantly to the intrinsic GTPase activity of both wild-type (WT) Rheb and the Y35A mutant. Furthermore, Asp65 was absolutely essential for the sensitivity of Rheb to the GAP activity of TSC2, whereas Gln64 was dispensable. Consistent with the in vitro data, expression of Rheb Y35A and D65A mutants in mammalian cells affected transduction of growth factor signals to mTORC1. Taken together, our observations reveal an efficient noncanonical mechanism of GTP hydrolysis by Rheb and suggest that autoinhibition of catalysis maintains Rheb in its highly activated state upon growth factor stimulation, which is necessary for the proper signal transduction to mTORC1. We previously showed that fluorescent-tagged nucleotides can alter the hydrolysis and exchange rates governing the GTPase cycle (Mazhab-Jafari et al., 2010Mazhab-Jafari M.T. Marshall C.B. Smith M. Gasmi-Seabrook G.M. Stambolic V. Rottapel R. Neel B.G. Ikura M. Real-time NMR study of three small GTPases reveals that fluorescent 2′(3′)-O-(N-methylanthraniloyl)-tagged nucleotides alter hydrolysis and exchange kinetics.J. Biol. Chem. 2010; 285: 5132-5136Crossref PubMed Scopus (34) Google Scholar). The most striking example we observed was that 2′-/3′-O-(N′-Methylanthraniloyl) (mant)GTP was hydrolyzed by Rheb ∼10-fold faster than GTP. This is not an intrinsic property of the modified nucleotide because the mant moiety inhibited GTP hydrolysis by RhoA and did not affect hydrolysis by Ras. The rate of mantGTP hydrolysis by Rheb is similar to that of Ras (Figure 1A), indicating that Rheb has a latent capacity for efficient catalysis. Interestingly, however, the rapid hydrolysis of mantGTP was independent of Rheb Gln64 (Mazhab-Jafari et al., 2010Mazhab-Jafari M.T. Marshall C.B. Smith M. Gasmi-Seabrook G.M. Stambolic V. Rottapel R. Neel B.G. Ikura M. Real-time NMR study of three small GTPases reveals that fluorescent 2′(3′)-O-(N-methylanthraniloyl)-tagged nucleotides alter hydrolysis and exchange kinetics.J. Biol. Chem. 2010; 285: 5132-5136Crossref PubMed Scopus (34) Google Scholar). The position of the fluorophore in a structure of Ras bound to a nonhydrolyzable analog of mantGTP (Scheidig et al., 1995Scheidig A.J. Franken S.M. Corrie J.E. Reid G.P. Wittinghofer A. Pai E.F. Goody R.S. X-ray crystal structure analysis of the catalytic domain of the oncogene product p21H-ras complexed with caged GTP and mant dGppNHp.J. Mol. Biol. 1995; 253: 132-150Crossref PubMed Scopus (58) Google Scholar) suggested that it may interact with the phenol ring of Tyr35 in switch I of Rheb. Remarkably, mutation of Tyr35 to Ala recapitulated the mant effect, increasing the rate of GTP hydrolysis by an order of magnitude (Figure 1B). Furthermore, the mant tag had no further effect on the catalytic activity of Rheb Y35A, suggesting that the mutation and the fluorophore stimulate hydrolysis through the same mechanism (Figure 1B). These observations indicate that Tyr35 autoinhibits the intrinsic GTPase activity of Rheb. We crystallized GDP-bound Rheb Y35A in the presence of excess GMPPNP (a nonhydrolyzable analog of GTP), and to our surprise, the asymmetric unit contained two molecules of Rheb: one bound to GDP and one to GMPPNP (Figures 2A–2C; Table 1). The overall protein fold is very similar to WT Rheb (Yu et al., 2005Yu Y. Li S. Xu X. Li Y. Guan K. Arnold E. Ding J. Structural basis for the unique biological function of small GTPase RHEB.J. Biol. Chem. 2005; 280: 17093-17100Crossref PubMed Scopus (62) Google Scholar) (backbone rmsd of 0.44 Å) with a few key differences. The nucleotide-binding pocket is completely solvent exposed in the GMPPNP-bound structure of Rheb Y35A, whereas in the WT protein the triphosphate group of the nucleotide is shielded from the solvent by the phenol ring of Tyr35, which forms a hydrogen bond with the γ-phosphate. In addition the γ-phosphate is 0.5 Å closer to Thr38 in the absence of Tyr35 (Figure 2D), which in the WT structure “pulls” the γ-phosphate toward the middle of switch I. Interestingly, the hydrolytic water is closer to the backbone carbonyl of Thr38 in the mutant (2.7 versus 3.8 Å in the WT protein) (Figure 2D), placing it in a more electron-rich environment that may enhance its polarization for an in-line nucleophilic attack to the γ-phosphate. It has been proposed that the corresponding backbone carbonyl of Ras (Thr35) contributes to the stabilization/activation of the catalytic water during intrinsic GTP hydrolysis (Buhrman et al., 2010Buhrman G. Holzapfel G. Fetics S. Mattos C. Allosteric modulation of Ras positions Q61 for a direct role in catalysis.Proc. Natl. Acad. Sci. USA. 2010; 107: 4931-4936Crossref PubMed Scopus (177) Google Scholar; Frech et al., 1994Frech M. Darden T.A. Pedersen L.G. Foley C.K. Charifson P.S. Anderson M.W. Wittinghofer A. Role of glutamine-61 in the hydrolysis of GTP by p21H-ras: an experimental and theoretical study.Biochemistry. 1994; 33: 3237-3244Crossref PubMed Scopus (112) Google Scholar). Comparison of our structure with that of WT Rheb indicates that Tyr35 pulls the γ-phosphate and catalytic water away from the Thr38 carbonyl, thus reducing its catalytic contribution.Table 1Data Collection and Refinement StatisticsRheb Y35AData CollectionSpace groupP 2 21 21Cell dimensions a, b, c (Å)57.2, 69.9, 79.2 α, β, γ (°)90, 90, 90Resolution (Å)46.4–2.0 (2.07–2.0)aData set was collected from one crystal.Rsym9.3 (41.2)I/σI20.7 (4.6)Completeness (%)99.7 (100)Redundancy7.0 (6.6)RefinementResolution (Å)26.9–2.0No. of reflections21,742Rwork/Rfree16.2/21.4No. of atoms Protein2,766 Ligand/Mg2+ ion62/2 Water252B factors Protein26.3 Ligand/Mg2+ ion21.6/24.2 Water30.3Rmsds Bond lengths (Å)0.007 Bond angles (°)1.14Ramachandran statistics Most favorable regions (%)96.7 Allowed regions (%)3.3 Disallowed regions (%)0Values in parentheses are for highest-resolution shell.a Data set was collected from one crystal. Open table in a new tab Values in parentheses are for highest-resolution shell. Switch I of Rheb Y35A does not undergo any substantial conformational change upon nucleotide hydrolysis, whereas this region of the WT protein exhibits a large structural change mediated by an interaction between Tyr35 and the γ-phosphate (Yu et al., 2005Yu Y. Li S. Xu X. Li Y. Guan K. Arnold E. Ding J. Structural basis for the unique biological function of small GTPase RHEB.J. Biol. Chem. 2005; 280: 17093-17100Crossref PubMed Scopus (62) Google Scholar) (Figures 2E and 2F). It was hypothesized that a similar nucleotide-dependent rearrangement of Rap Tyr32 would be energetically unfavorable to the GTPase reaction (Cherfils et al., 1997Cherfils J. Ménétrey J. Le Bras G. Janoueix-Lerosey I. de Gunzburg J. Garel J.R. Auzat I. Crystal structures of the small G protein Rap2A in complex with its substrate GTP, with GDP and with GTPgammaS.EMBO J. 1997; 16: 5582-5591Crossref PubMed Scopus (61) Google Scholar), consistent with our observation that nucleotide hydrolysis is accelerated by a mutation that disrupts this conformational change. Previous work has shown that Gln64, corresponding to the catalytic Gln61 of Ras, is not involved in GTP hydrolysis by WT Rheb (Inoki et al., 2003Inoki K. Li Y. Xu T. Guan K.L. Rheb GTPase is a direct target of TSC2 GAP activity and regulates mTOR signaling.Genes Dev. 2003; 17: 1829-1834Crossref PubMed Scopus (1418) Google Scholar; Li et al., 2004Li Y. Inoki K. Guan K.L. Biochemical and functional characterizations of small GTPase Rheb and TSC2 GAP activity.Mol. Cell. Biol. 2004; 24: 7965-7975Crossref PubMed Scopus (188) Google Scholar; Marshall et al., 2009Marshall C.B. Ho J. Buerger C. Plevin M.J. Li G.Y. Li Z. Ikura M. Stambolic V. Characterization of the intrinsic and TSC2-GAP-regulated GTPase activity of Rheb by real-time NMR.Sci. Signal. 2009; 2: ra3Crossref PubMed Scopus (48) Google Scholar). Likewise, Gln64 remains in a noncatalytic conformation in the structure of Rheb Y35A (see Figure S1A available online) and is not required for the accelerated hydrolysis of GTP by Rheb Y35A (Figures S1D and S2A). Because the catalytic residues of other small GTPase superfamily members are found in the N terminus of switch II, we examined this region for residues with electronegative side chains that may contribute to the hydrolytic reaction. Immediately downstream of Gln64 are two residues with acidic side chains: Asp65 and Glu66 (Figure 2G). The crystal structure of Rheb Y35A shows that the backbone of the N-terminal loop of switch II of this mutant is displaced by an average of 1 Å toward the nucleotide-binding pocket relative to the WT, which brings the side-chain carboxylate of Asp65 closer to the nucleotide by 1 Å: average Asp65Oδ1,2WT − average Asp65Oδ1,2Y35A (Figure 2G). Mutation of Asp65 to Ala reduced the intrinsic hydrolysis of Rheb Y35A by more than 60% and that of WT by 30% (Figure 3A), as did the conservative substitution of Asp65 by Asn (Figure S2B). On the other hand, mutations of Glu66 had no effect on intrinsic GTPase activity (Figure S2C), consistent with its perpendicular orientation away from the nucleotide (Figure 2G). We also tested all other residues found within 10 Å of the hydrolytic water in the Rheb Y35A structure that could potentially provide (1) a negative charge to activate this water molecule, or (2) a positive charge to stabilize the β- and γ-phosphates in the transition state for hydrolysis (Figure S1). There was no change in the rate of intrinsic nucleotide hydrolysis associated with R15G, S16A, or D36A mutations (Figures S1B, S1C, and S1E). The only other charged residues within 10 Å of the hydrolytic water are Lys19 and Asp60 of the highly conserved G1 and G3 box motifs, respectively. The Rheb K19A mutant failed to express, presumably due to impaired nucleotide binding, and D60A was highly unstable and could not be loaded with GTP, consistent with the role of this residue in Mg2+ coordination (Yu et al., 2005Yu Y. Li S. Xu X. Li Y. Guan K. Arnold E. Ding J. Structural basis for the unique biological function of small GTPase RHEB.J. Biol. Chem. 2005; 280: 17093-17100Crossref PubMed Scopus (62) Google Scholar). These data strongly suggest that Asp65 is the sole candidate for a catalytic residue in Rheb. Notably, carboxylates are more potent nucleophiles than carboxamides, and consistently, the Q61E substitution increased the GTPase activity of Ras (Frech et al., 1994Frech M. Darden T.A. Pedersen L.G. Foley C.K. Charifson P.S. Anderson M.W. Wittinghofer A. Role of glutamine-61 in the hydrolysis of GTP by p21H-ras: an experimental and theoretical study.Biochemistry. 1994; 33: 3237-3244Crossref PubMed Scopus (112) Google Scholar). In the structure of WT Rheb, the carboxylate of Asp65 is 12 Å (average Asp65Oδ1,2) from the γ-phosphate in a single conformation, whereas the electron density of Rheb Y35A indicates that Asp65 exists in two conformations, 11.0 and 12.0 Å from the γ-phosphate, respectively (Figure S1F). By comparison the catalytic carboxamide of Ras (Gln61Oε) has been found at distances varying from 4.7 to 12.2 Å from the γ-phosphate (median distance of 8.1Å) (Figure S3A) in available crystallographic snapshots, consistent with the dynamic nature of switch II determined by NMR studies (Ito et al., 1997Ito Y. Yamasaki K. Iwahara J. Terada T. Kamiya A. Shirouzu M. Muto Y. Kawai G. Yokoyama S. Laue E.D. et al.Regional polysterism in the GTP-bound form of the human c-Ha-Ras protein.Biochemistry. 1997; 36: 9109-9119Crossref PubMed Scopus (142) Google Scholar). Thus, despite its established role as a catalytic residue (Frech et al., 1994Frech M. Darden T.A. Pedersen L.G. Foley C.K. Charifson P.S. Anderson M.W. Wittinghofer A. Role of glutamine-61 in the hydrolysis of GTP by p21H-ras: an experimental and theoretical study.Biochemistry. 1994; 33: 3237-3244Crossref PubMed Scopus (112) Google Scholar), Gln61 is rarely found in a catalytically competent conformation in Ras crystal structures, presumably because this state is transient and energetically unfavorable (Fraser et al., 2011Fraser J.S. van den Bedem H. Samelson A.J. Lang P.T. Holton J.M. Echols N. Alber T. Accessing protein conformational ensembles using room-temperature X-ray crystallography.Proc. Natl. Acad. Sci. USA. 2011; 108: 16247-16252Crossref PubMed Scopus (383) Google Scholar; Grant et al., 2009Grant B.J. Gorfe A.A. McCammon J.A. Ras conformational switching: simulating nucleotide-dependent conformational transitions with accelerated molecular dynamics.PLoS Comput. Biol. 2009; 5: e1000325Crossref PubMed Scopus (146) Google Scholar). Similarly, our Y35A structure and the previous WT Rheb structure (Yu et al., 2005Yu Y. Li S. Xu X. Li Y. Guan K. Arnold E. Ding J. Structural basis for the unique biological function of small GTPase RHEB.J. Biol. Chem. 2005; 280: 17093-17100Crossref PubMed Scopus (62) Google Scholar) both appear to be energetically stable states, with the conformations of Asp65 stabilized primarily by ionic and polar interactions with the Arg15 and Ser68 side chains, which are also found in two alternate conformations in our structure (Figure S1F). Interestingly, comparison of WT and Y35A 1H-15N heteronuclear single-quantum coherence (HSQC) spectra revealed increased line broadening for residues in the P loop and the N terminus of switch II of GTP-bound Rheb Y35A (Figures 2H and S4A), suggesting elevated dynamics in μs-ms timescale. This could allow the N terminus of switch II to sample alternate conformations closer to the nucleotide and the catalytic water. The elevated dynamics of the N-terminal region of switch II and its proximity to the nucleotide-binding site in Rheb Y35A is consistent with the greater impact on catalysis of Asp65 mutations in the Y35A mutant than in WT Rheb (Figure 3A). Hence, in addition to affecting the orientation of the nucleotide and hydrolytic water, Tyr35 may reduce the intrinsic GTPase activity of Rheb by restricting the dynamics of switch II and displacing it from the nucleotide-binding site. Relative to Ras, the catalytic Gln residues of Rho subfamily GTPases were found closer to the γ-phosphate (median distance of 5.5Å) (Figures S3A and S3C), which may contribute to their faster intrinsic nucleotide hydrolysis rate (Mazhab-Jafari et al., 2010Mazhab-Jafari M.T. Marshall C.B. Smith M. Gasmi-Seabrook G.M. Stambolic V. Rottapel R. Neel B.G. Ikura M. Real-time NMR study of three small GTPases reveals that fluorescent 2′(3′)-O-(N-methylanthraniloyl)-tagged nucleotides alter hydrolysis and exchange kinetics.J. Biol. Chem. 2010; 285: 5132-5136Crossref PubMed Scopus (34) Google Scholar). On the other hand, Gln63, which was recently proposed to be a noncanonical catalytic residue of Rap in GAP1IP4BP-mediated GTP hydrolysis (Sot et al., 2010Sot B. Kötting C. Deaconescu D. Suveyzdis Y. Gerwert K. Wittinghofer A. Unravelling the mechanism of dual-specificity GAPs.EMBO J. 2010; 29: 1205-1214Crossref PubMed Scopus (42) Google Scholar), is found with a median distance of 11.8 Å from the γ-phosphate in structures of free Rap (Figure S3A), consistent with the slow nucleotide hydrolysis of this GTPase. In Ras, Gly12, Gly13, and Gln61 are the major sites of oncogenic mutations. Mutation of Ras Gly12 to any other residue hinders GTP hydrolysis by sterically occluding access of the catalytic residue Gln61 to the hydrolytic water and nucleotide (Krengel et al., 1990Krengel U. Schlichting I. Scherer A. Schumann R. Frech M. John J. Kabsch W. Pai E.F. Wittinghofer A. Three-dimensional structures of H-ras p21 mutants: molecular basis for their inability to function as signal switch molecules.Cell. 1990; 62: 539-548Abstract Full Text PDF PubMed Scopus (325) Google Scholar). However, Rheb has an Arg in this position, and its mutation to Gly (R15G) does not increase the catalytic activity of Rheb Y35A (Figure S1E) or WT (Im et al., 2002Im E. von Lintig F.C. Chen J. Zhuang S. Qui W. Chowdhury S. Worley P.F. Boss G.R. Pilz R.B. Rheb is in a high activation state and inhibits B-Raf kinase in mammalian cells.Oncogene. 2002; 21: 6356-6365Crossref PubMed Scopus (116) Google Scholar; Li et al., 2004Li Y. Inoki K. Guan K.L. Biochemical and functional characterizations of small GTPase Rheb and TSC2 GAP activity.Mol. Cell. Biol. 2004; 24: 7965-7975Crossref PubMed Scopus (188) Google Scholar; Marshall et al., 2009Marshall C.B. Ho J. Buerger C. Plevin M.J. Li G.Y. Li Z. Ikura M. Stambolic V. Characterization of the intrinsic and TSC2-GAP-regulated GTPase activity of Rheb by real-time NMR.Sci. Signal. 2009; 2: ra3Crossref PubMed Scopus (48) Google Scholar; Yamagata et al., 1994Yamagata K. Sanders L.K. Kaufmann W.E. Yee W. Barnes C.A. Nathans D. Worley P.F. rheb, a growth factor- and synaptic activity-regulated gene, encodes a novel Ras-related protein.J. Biol. Chem. 1994; 269: 16333-16339Abstract Full Text PDF PubMed Google Scholar). The distinctive impact of P loop residues on the activities of Ras and Rheb lends further support to the different molecular mechanisms of action of these two closely related GTPase homologs. Mutation of the solvent-exposed residue Asp65 to Ala (D65A) did not perturb the structure of Rheb, on the basis of minimal and localized chemical shift perturbations in the 1H-15N HSQC spectra that were mainly confined to switch II (Figure S5), but totally abolished the susceptibility of Rheb to the GAP activity of TSC2 (Figure 3B). Furthermore, even conservative mutations of Asp65 (D65E/N) rendered Rheb totally insensitive to the activity of TSC2GAP. The strict requirement for the geometry and charge of this side chain suggest that it might be a critical catalytic residue for the GAP-mediated hydrolysis reaction. We also tested the sensitivity of the GTPase activity of Rheb Y35A to the action of TSC2GAP and found that the GTPase activity of this mutant was not further stimulated by the addition of the GAP domain of TSC2 (Figure 4A). An analogous mutation (Y32A) impaired the sensitivity of Rap GTPase to the function of RapGAP (Brinkmann et al., 2002Brinkmann T. Daumke O. Herbrand U. Kühlmann D. Stege P. Ahmadian M.R. Wittinghofer A. Rap-specific GTPase activating protein follows an alternative mechanism.J. Biol. Chem. 2002; 277: 12525-12531Crossref PubMed Scopus (65) Google Scholar; Scrima et al., 2008Scrima A. Thomas C. Deaconescu D. Wittinghofer A. The Rap-RapGAP complex: GTP hydrolysis without catalytic glutamine and arginine residues.EMBO J. 2008; 27: 1145-1153Crossref PubMed Scopus (91) Google Scholar); however, a conservative mutation (Y32F) was tolerated. Interestingly, the Y35F mutation was sufficient to render Rheb insensitive to the function of the TSC2GAP (Figure 4B), highlighting differences in the details of molecular recognition in these two homologous systems. To better understand the energetic basis of Tyr35 autoinhibition, we analyzed the thermodynamics of the GTP hydrolysis reaction using an Arrhenius plot (Figure S6). This powerful technique allows one to extract energetic parameters, such as enthalpy, entropy, and free energy, from the highly unstable and low-populated transition state of an enzymatic reaction. The increased catalytic activity of Rheb Y35A was associated with a large reduction in the activation enthalpy for GTP hydrolysis (Figure S6; Table 2). However, the activation entropy was also r" @default.
• W2150953043 created "2016-06-24" @default.
• W2150953043 creator A5016424930 @default.
• W2150953043 creator A5023356870 @default.
• W2150953043 creator A5025568767 @default.
• W2150953043 creator A5027951205 @default.
• W2150953043 creator A5054067261 @default.
• W2150953043 creator A5079223053 @default.
• W2150953043 creator A5079720198 @default.
• W2150953043 date "2012-09-01" @default.
• W2150953043 modified "2023-09-24" @default.
• W2150953043 title "An Autoinhibited Noncanonical Mechanism of GTP Hydrolysis by Rheb Maintains mTORC1 Homeostasis" @default.
• W2150953043 cites W1515372146 @default.
• W2150953043 cites W1539796472 @default.
• W2150953043 cites W1968453436 @default.
• W2150953043 cites W1969319225 @default.
• W2150953043 cites W1969771018 @default.
• W2150953043 cites W1976371008 @default.
• W2150953043 cites W1982660730 @default.
• W2150953043 cites W1984412362 @default.
• W2150953043 cites W1992864103 @default.
• W2150953043 cites W1993735775 @default.
• W2150953043 cites W2006176820 @default.
• W2150953043 cites W2031178514 @default.
• W2150953043 cites W2033204562 @default.
• W2150953043 cites W2033498369 @default.
• W2150953043 cites W2036277985 @default.
• W2150953043 cites W2036935837 @default.
• W2150953043 cites W2038989070 @default.
• W2150953043 cites W2041781518 @default.
• W2150953043 cites W2044247583 @default.
• W2150953043 cites W2045016256 @default.
• W2150953043 cites W2051728948 @default.
• W2150953043 cites W2056137479 @default.
• W2150953043 cites W2056668858 @default.
• W2150953043 cites W2056739666 @default.
• W2150953043 cites W2058834594 @default.
• W2150953043 cites W2059172461 @default.
• W2150953043 cites W2061196778 @default.
• W2150953043 cites W2063354318 @default.
• W2150953043 cites W2063484479 @default.
• W2150953043 cites W2082570884 @default.
• W2150953043 cites W2082853003 @default.
• W2150953043 cites W2085248799 @default.
• W2150953043 cites W2087432921 @default.
• W2150953043 cites W2099187439 @default.
• W2150953043 cites W2104235973 @default.
• W2150953043 cites W2109263914 @default.
• W2150953043 cites W2110656190 @default.
• W2150953043 cites W2112078762 @default.
• W2150953043 cites W2116332105 @default.
• W2150953043 cites W2124887866 @default.
• W2150953043 cites W2132428389 @default.
• W2150953043 cites W2139112892 @default.
• W2150953043 cites W2144081223 @default.
• W2150953043 cites W2153552867 @default.
• W2150953043 cites W2169821755 @default.
• W2150953043 cites W2180229411 @default.
• W2150953043 doi "https://doi.org/10.1016/j.str.2012.06.013" @default.
• W2150953043 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/22819219" @default.
• W2150953043 hasPublicationYear "2012" @default.
• W2150953043 type Work @default.
• W2150953043 sameAs 2150953043 @default.
• W2150953043 citedByCount "29" @default.
• W2150953043 countsByYear W21509530432013 @default.
• W2150953043 countsByYear W21509530432014 @default.
• W2150953043 countsByYear W21509530432015 @default.
• W2150953043 countsByYear W21509530432016 @default.
• W2150953043 countsByYear W21509530432017 @default.
• W2150953043 countsByYear W21509530432018 @default.
• W2150953043 countsByYear W21509530432020 @default.
• W2150953043 countsByYear W21509530432021 @default.
• W2150953043 crossrefType "journal-article" @default.
• W2150953043 hasAuthorship W2150953043A5016424930 @default.
• W2150953043 hasAuthorship W2150953043A5023356870 @default.
• W2150953043 hasAuthorship W2150953043A5025568767 @default.
• W2150953043 hasAuthorship W2150953043A5027951205 @default.
• W2150953043 hasAuthorship W2150953043A5054067261 @default.
• W2150953043 hasAuthorship W2150953043A5079223053 @default.
• W2150953043 hasAuthorship W2150953043A5079720198 @default.
• W2150953043 hasBestOaLocation W21509530431 @default.
• W2150953043 hasConcept C111472728 @default.
• W2150953043 hasConcept C118716 @default.
• W2150953043 hasConcept C11960822 @default.
• W2150953043 hasConcept C138885662 @default.
• W2150953043 hasConcept C181199279 @default.
• W2150953043 hasConcept C185592680 @default.
• W2150953043 hasConcept C207332259 @default.
• W2150953043 hasConcept C2778937405 @default.
• W2150953043 hasConcept C55493867 @default.
• W2150953043 hasConcept C63645605 @default.
• W2150953043 hasConcept C75217442 @default.
• W2150953043 hasConcept C86803240 @default.
• W2150953043 hasConcept C89611455 @default.
• W2150953043 hasConcept C95444343 @default.
• W2150953043 hasConcept C98490376 @default.
• W2150953043 hasConceptScore W2150953043C111472728 @default.
• W2150953043 hasConceptScore W2150953043C118716 @default.

• next