Structural Basis for the Unique Biological Function of Small GTPase RHEB

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′-(β,γ-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 α-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. 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′-(β,γ-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 α-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. 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 (1Yamagata K. Sanders L.K. Kaufmann W.E. Yee W. Barnes C.A. Nathans D. Worley P.F. J. Biol. Chem. 1994; 269: 16333-16339Abstract Full Text PDF PubMed Google Scholar, 2Mizuki N. Kimura M. Ohno S. Miyata S. Sato M. Ando H. Ishihara M. Goto K. Watanabe S. Yamazaki M. Ono A. Taguchi S. Okumura K. Nogami M. Taguchi T. Ando A. Inoko H. Genomics. 1996; 34: 114-118Crossref PubMed Scopus (27) Google Scholar, 3Aspuria P-J. Tamanoi F. Cell. Signal. 2004; 16: 1105-1112Crossref PubMed Scopus (166) Google Scholar). 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 TSC1 1The abbreviations used are: TSC, tuberous sclerosis complex; GAP, GTPase-activating protein; TSC2GAP, GAP domain of TSC2; mTOR, mammalian target of rapamycin; GppNHp, 5′-(β,γ-imide)triphosphate; r.m.s., root mean square. and TSC2 (tuberous sclerosis complex 1 and 2) and the mammalian target of rapamycin (mTOR) to stimulate cell growth (4Gao X. Zhang Y. Arrazola P. Hino O. Kobayashi T. Yeung R.S. Ru B. Pan D. Nat. Cell Biol. 2002; 4: 699-704Crossref PubMed Scopus (575) Google Scholar, 5Inoki K. Li Y. Zhu T. Wu J. Guan K.L. Nat. Cell Biol. 2002; 4: 648-657Crossref PubMed Scopus (2406) Google Scholar, 6Manning B.D. Tee A.R. Logsdon M.N. Blenis J. Cantley L.C. Mol. Cell. 2002; 10: 151-162Abstract Full Text Full Text PDF PubMed Scopus (1278) Google Scholar, 7Tee A.R. Fingar D.C. 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Tippett P. Edwards J.H. Kwiatkowski D.J. Super M. Mueller R. Fryer A. Clarke A. Webb D. Osborne J. Ann. Hum. Genet. 1994; 58: 107-127Crossref PubMed Scopus (234) Google Scholar, 20Maheshwar M.M. Cheadle J.P. Jones A.C. Myring J. Fryer A.E. Harris P.C. Sampson J.R. Hum. Mol. Genet. 1997; 6: 1991-1996Crossref PubMed Scopus (123) Google Scholar). 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 (21Gomez M.R. Tuberous Sclerosis. 3rd Ed. Oxford University Press, Oxford1999Google Scholar, 22Sparagana S.P. Roach E.S. Curr. 13: PubMed Scopus Google Scholar). TSC1 and TSC2 a and complex that the of Rheb. and 1 and protein TSC2 a conserved GTPase-activating protein domain its which is to M.M. R. M. Cheadle J.P. B. M. Sampson J.R. Hum. Mol. Genet. 1996; 5: PubMed Scopus Google Scholar). Rheb by its GTPase activity A.F. Rebhun J.F. Clark G.J. Quilliam L.A. J. Biol. Chem. 2003; 278: 32493-32496Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar, 9Garami A. Zwartkruis F.J. Nobukuni T. Joaquin M. Roccio M. Stocker H. Kozma S.C. Hafen E. Bos J.L. Thomas G. Mol. Cell. 2003; 11: 1457-1466Abstract Full Text Full Text PDF PubMed Scopus (850) Google Scholar, 10Inoki K. Li Y. Xu T. Guan K.L. Genes Dev. 2003; 17: 1829-1834Crossref PubMed Scopus (1424) Google Scholar, 16Tee A.R. Manning B.D. Roux P.P. Cantley L.C. Blenis J. Curr. Biol. 2003; 13: 1259-1268Abstract Full Text Full Text PDF PubMed Scopus (947) Google Scholar, 17Zhang Y. Gao X. Saucedo L.J. Ru B. Edgar B.A. Pan D. Nat. Cell Biol. 2003; 5: 578-581Crossref PubMed Scopus (716) Google Scholar, Y. Inoki K. Guan K.L. Mol. Cell. Biol. 2004; PubMed Scopus Google Scholar). The is a of cell growth in to growth and Rheb the of 1 and protein 1 of Mutations in Rheb can cause cell growth of Rheb can an in cell and the cell of of TSC2 identified in TSC are in the of 1 and protein 1 K. Li Y. Zhu T. Wu J. Guan K.L. Nat. Cell Biol. 2002; 4: 648-657Crossref PubMed Scopus (2406) Google Scholar, 20Maheshwar M.M. Cheadle J.P. Jones A.C. Myring J. Fryer A.E. Harris P.C. Sampson J.R. Hum. Mol. Genet. 1997; 6: 1991-1996Crossref PubMed Scopus (123) Google Scholar, Y. Inoki K. Guan K.L. Mol. Cell. Biol. 2004; PubMed Scopus Google that the of protein be of the of the mechanism of by Rheb is Rheb with other small of the Ras/Rap subfamily and to to the Ras/Rap subfamily (1Yamagata K. Sanders L.K. Kaufmann W.E. Yee W. Barnes C.A. Nathans D. Worley P.F. J. Biol. Chem. 1994; 269: 16333-16339Abstract Full Text PDF PubMed Google Scholar). Rheb unique biological properties that substantially from of other of the Ras/Rap small the of GTP hydrolysis the of a conserved the catalytic of GTPase to Gln61 in Ras), the insertion of an arginine finger of GAP the catalytic site, and the conformational and of the switch of the GTPase K. J.F. S.J. S.J. 1997; PubMed Scopus Google Scholar, K. M.R. W. L. A. F. A. 1997; PubMed Scopus Google Scholar, A. PubMed Scopus Google Scholar). Rheb a conserved the its to the GTPase activity of Rheb K. Li Y. Xu T. Guan K.L. Genes Dev. 2003; 17: 1829-1834Crossref PubMed Scopus (1424) Google Scholar, Y. Inoki K. Guan K.L. Mol. Cell. Biol. 2004; PubMed Scopus Google and the Rheb functions to the Rheb to the G.J. K. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). the Ras, which is to the of the Rheb is to Y. Inoki K. Guan K.L. Mol. Cell. Biol. 2004; PubMed Scopus Google Scholar). both and studies have identified an arginine finger in Y. Inoki K. Guan K.L. Mol. Cell. Biol. 2004; PubMed Scopus Google Scholar, O. M. P.P. A. 2004; PubMed Scopus Google Scholar). Rheb a low intrinsic GTPase activity and in a E. J. S. W. S. Worley P.F. 2002; PubMed Scopus Google Scholar). distinct properties Rheb a unique in the the biological function(s) of Rheb in and the potential of Rheb with its and we structural studies of human Rheb of with a molecular of N. Kimura M. Ohno S. Miyata S. Sato M. Ando H. Ishihara M. Goto K. Watanabe S. Yamazaki M. Ono A. Taguchi S. Okumura K. Nogami M. Taguchi T. Ando A. Inoko H. Genomics. 1996; 34: 114-118Crossref PubMed Scopus (27) Google Scholar). The the GTPase the are with a and a conserved that important in the of Rheb and its with A.F. Rebhun J.F. Clark G.J. Quilliam L.A. J. Biol. Chem. 2003; 278: 32493-32496Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar, 10Inoki K. Li Y. Xu T. Guan K.L. Genes Dev. 2003; 17: 1829-1834Crossref PubMed Scopus (1424) Google Scholar). We report here the structures of the GTPase domain of in complexes with GDP, GTP, and a GTP GppNHp, and of Rheb with other small GTPases novel structural features of Rheb and a molecular basis for its unique biological properties. This is in the biological of Rheb in the and provide for the of novel for TSC and other and and to the structural studies of other small we a of to as that the to and of and the and data of in complexes with GDP, GTP, and GppNHp are Y. Y. Li S. H. J. D. 2004; PubMed Scopus Google Scholar). of the data used for and of the complexes are in of data and data in to the of of and and in to the in a new and of the complex was the molecular as in the P. J. M. T. D. PubMed Scopus Google with the structures of small human and and J. J. G. J. J.R. J. 1997; 16: PubMed Scopus Google Scholar, N. G. A. F. A. PubMed Scopus Google as the The structures of the and complexes the molecular with the of the complex as the was the with of the for the the that GppNHp or and an in both switch I and II and in in different the in a of as in in to or was with the M. A. PubMed Scopus Google and by and and of structures of the GTPase domain of in complexes with GDP, GTP, and GppNHp and of the and the is in 1 the switch II in different The of that of other small GTPases of the different complexes are to of the complex the and complexes an of and and of the complex the complex an of of and its with other small of the The of are of human Ras, that of is by a I and II are in and the is in The GppNHp is as a as a and the conserved as of with the conformational in the switch I and II The complex is in the complex in the complex in and the complex in The is in the GppNHp and the in the complex are of with other small GTPases from human with The of the structures used in are in are in and conserved are in The structures of and are the Rheb with and of the Ras/Rap subfamily and with other small GTPases the Ras/Rap thus the of Rheb II and between and small that is to and to and of with small GTPases an in the of The structural different complexes and between and the other small GTPases in the switch I and switch II that are in and with and A. PubMed Scopus Google Scholar, A. A. Oxford University Press, Oxford1999Google Scholar). the switch of are as to of Ras, and to of and structural of with other small or of in of in in a new the the of a GTP, or and an which is for the GTPase the catalytic The and with the of have also in other small GTPases in complexes with GTP, GTP or the the is by in a from the and of the and and and and 2 and conserved that is to the the of the in GTP hydrolysis was by with the chain of and and and the of GppNHp and with and The of GppNHp with conserved Tyr35 and The of the shielded by switch I, by the side chain of change was in the and conformation of the and the conformation of the protein between the complex and the the the by the and was by the of the of the and in a the by of GTP and the by the of the conformation of switch I the was to and the space by switch I in the complex and with the phosphate and of of Rheb in GTP of with other small GTPases that the a different small GTPases and between and forms of between is in the of to 2 and of the Ras/Rap subfamily a conserved in the to in of to other residue the intrinsic GTPase activity of Ras, making the K. M.R. W. L. A. F. A. 1997; PubMed Scopus Google Scholar, M. Biochem. PubMed Scopus Google Scholar, U. L. A. R. M. J. W. A. Cell. Full Text PDF PubMed Scopus Google Scholar). the intrinsic GTPase activity and is to by This with GTP hydrolysis by steric hindrance between the side chain of and the side chain of Gln61 a conformation U. L. A. R. M. J. W. A. Cell. Full Text PDF PubMed Scopus Google Scholar). Rheb a conserved the and the Rheb low intrinsic GTPase activity and a GTP which is that of Ras), leading to the that of Rheb is for properties E. J. S. W. S. Worley P.F. 2002; PubMed Scopus Google Scholar). the Rheb can be by A.F. Rebhun J.F. Clark G.J. Quilliam L.A. J. Biol. Chem. 2003; 278: 32493-32496Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar, 9Garami A. Zwartkruis F.J. Nobukuni T. Joaquin M. Roccio M. Stocker H. Kozma S.C. Hafen E. Bos J.L. Thomas G. Mol. Cell. 2003; 11: 1457-1466Abstract Full Text Full Text PDF PubMed Scopus (850) Google Scholar, 10Inoki K. 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Worley P.F. 2002; PubMed Scopus Google Scholar). the and the side chain of was and of the side chain of side chain a with the side chain of and its a with the chain of of the of switch II and the displacement of Gln64 the side chain of have steric hindrance with the side chain of as seen between and Gln61 in or with the and the the side chain was from switch and its with and the of a with the of both the side chain of was in with either the or with that is in GTP hydrolysis is for its GTP it have a different in GTP hydrolysis. biochemical data have that the Rheb is to by that a in the of Rheb with Y. Inoki K. Guan K.L. Mol. Cell. Biol. 2004; PubMed Scopus Google Scholar). I GTP/GDP I of small GTPases important in and with and the in and structures of switch I the of small GTPases to to other small switch I of with a for the protein in the in the and in the that switch I of is to of and to of and of the in switch I between Rheb and and of the conserved different the other of the between Rheb and and was between Rheb and also that switch I of is to of and from of and in complexes with GDP, GTP, or other small switch I in the conformational change with the or the of the Tyr35 and the of the the conformational change was in and between the and the the conformational of switch I between and Ras/Rap in complexes with in complexes with GTP or GppNHp, the of switch I of from the of by and to in the complex by in the The side of in and in have to the for N. G. R. A. Nat. Biol. 1996; PubMed Scopus Google Scholar). The was by a and residue in that Rheb a different from that of Ras/Rap with its in a different II a and a between and other small GTPases in the switch II 2 and small switch II a α-helical conformation to in in complexes with GTP or GppNHp in complexes with with the to A. A. Oxford University Press, Oxford1999Google Scholar). During the GTP/GDP cycling, switch II conformational in the structures of complexes with GTP and GppNHp, switch II an conformation instead of the α-helical conformation, and the with a low for the protein in the complex and in the 2 and a an extended The was the and the of and and with structural The was to and with the of the to the unique switch II conformation of Rheb. the of the was from the residue in or Gln64 was by with the residue Gln61 in or in The side chain of Gln64 was from the and in a by and with GTP the or other the catalytic switch II a conformation with and a low in the complex for the and conformational the of GTP/GDP 2 and between the and forms was a of the chain group of in of the chain of the and a displacement of in The side chain of Gln64 was and with the chain group of and the side of and the side chain of was also and its with the side chain of and the chain of was the conformational change of the of switch the in the complex was by 2 with the or and to making the with The different complexes with GTP, GppNHp, and with different space group and different Y. Y. Li S. H. J. D. 2004; PubMed Scopus Google Scholar). of that a of switch II in it is that the unique conformation of switch II is by it be an of and to its biological II for the GTPase of biochemical and cell biological data have that Rheb a low intrinsic GTPase activity and a GTP leading to the that the Rheb as a protein G.J. K. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, E. J. S. W. S. Worley P.F. 2002; PubMed Scopus Google Scholar). structural data that was in GTP hydrolysis and was for the low GTPase activity of Rheb is the molecular basis of the low GTPase activity of small GTPases a conserved residue in switch to Gln61 of and Gln64 of Gln61 of is in GTP hydrolysis by a conserved that the of GTP K. M.R. W. L. A. F. A. 1997; PubMed Scopus Google Scholar, U. R.S. W. A. J. PubMed Scopus Google Scholar). of Gln61 in a of the GTP and to a of the distinct switch II conformation in the the chain of Gln64 was by from its in Ras, and its side chain was a and with the or the important the catalytic Gln64 of is to in GTP which is with the biochemical data that the have a the GTPase activity of Rheb K. Li Y. Xu T. Guan K.L. Genes Dev. 2003; 17: 1829-1834Crossref PubMed Scopus (1424) Google Scholar, Y. Inoki K. Guan K.L. Mol. Cell. Biol. 2004; PubMed Scopus Google Scholar). the Rheb is to Y. Inoki K. Guan K.L. Mol. Cell. Biol. 2004; PubMed Scopus Google Scholar). together, data to propose that the unique switch II conformation and the of Gln64 to in GTP hydrolysis are for the low GTPase activity of Rheb and for its GTP-binding of a to residue identified in or Y. Inoki K. Guan K.L. Mol. Cell. Biol. 2004; PubMed Scopus Google Scholar, O. M. P.P. A. 2004; PubMed Scopus Google Scholar). conserved of are to be for its GAP activity are to the arginine finger of Y. Inoki K. Guan K.L. Mol. Cell. Biol. 2004; PubMed Scopus Google Scholar). Rheb and an arginine finger from The structures of provide a potential for the conformation of switch I in was to that of other small GTPases in complexes with GTP or GppNHp, was a in the conformation of a conserved in to Tyr35 of of and of the of the the side chain of was from the and with GppNHp, and is an to the phosphate of the U. R.S. W. A. J. PubMed Scopus Google the of the a conformation, and the arginine finger of the catalytic of the and forms with the phosphate of GTP K. M.R. W. L. A. F. A. 1997; PubMed Scopus Google Scholar). in the structures of Rheb in complexes with GTP or GppNHp, Tyr35 was to the its side chain by to the of the phosphate of GTP with and its a with the of GTP the or of a conformation as Tyr35 of Rheb. The of Tyr35 of Rheb or of the phosphate moiety of GTP to the closure of the GTP-binding site, which the from also the of a potential arginine finger to the phosphate of the This conformation of the GTP-binding be an of Rheb and and the molecular basis of arginine finger is from it be that an residue of a as that of the arginine finger of to the the phosphate of The switch I of small GTPases and the conserved residue was to it is that the GTP-binding in Rheb to a potential arginine finger to the catalytic GTP the of GTP hydrolysis GAP the GTP hydrolysis of small GTPase by an arginine finger K. J.F. S.J. S.J. 1997; PubMed Scopus Google Scholar, K. M.R. W. L. A. F. A. 1997; PubMed Scopus Google Scholar). The side chain of forms with the of GTP to the and its chain group forms a with the side chain of the catalytic Gln61 of to the with the The of GAP with also conformational that the switch and a the of the hydrolysis Gln64 of Rheb appears to be and appears to conserved arginine residue to the arginine finger of The structures of Rheb provide a molecular basis for Gln64 of Rheb is in GTP hydrolysis and arginine finger is from stimulate GTP hydrolysis of and structural data have that uses a catalytic to stimulate GTP hydrolysis of O. M. P.P. A. 2004; PubMed Scopus Google Scholar). an residue and of residue its GAP activity Rheb A. Zwartkruis F.J. Nobukuni T. Joaquin M. Roccio M. Stocker H. Kozma S.C. Hafen E. Bos J.L. Thomas G. Mol. Cell. 2003; 11: 1457-1466Abstract Full Text Full Text PDF PubMed Scopus (850) Google Scholar, 17Zhang Y. Gao X. Saucedo L.J. Ru B. Edgar B.A. Pan D. Nat. Cell Biol. 2003; 5: 578-581Crossref PubMed Scopus (716) Google Scholar, Y. Inoki K. Guan K.L. Mol. Cell. Biol. 2004; PubMed Scopus Google Scholar). is that Asn1643 of as the catalytic residue in the GTP hydrolysis of Rheb in a as of other small GTPases and of with conformational of both proteins and the and the switch of Rheb to the GTP hydrolysis conformational be to Gln64 the we a potential of Gln64 of Rheb and an mechanism of GTP hydrolysis in the of of Rheb in complex with reveal the between and the potential conformational of both proteins and provide the catalytic mechanism of GTP hydrolysis of Rheb. Rheb with and of the Ras/Rap subfamily and with other small and of the subfamily and of the structural data that Rheb is to and to and and to the Ras/Rap which is with the results (1Yamagata K. Sanders L.K. Kaufmann W.E. Yee W. Barnes C.A. Nathans D. Worley P.F. J. Biol. Chem. 1994; 269: 16333-16339Abstract Full Text PDF PubMed Google Scholar, Curr. Cell Biol. PubMed Scopus Google Scholar). Rheb distinct biological and biochemical properties and unique structural in the important that are substantially different from other of the Ras/Rap Taking the genetic, biochemical, biological, and structural data together, we propose that Rheb forms a new group of the Ras/Rap subfamily and a novel GTP hydrolysis mechanism that utilizes Asn1643 of instead of Gln64 of Rheb as the catalytic residue in GTP hydrolysis.