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• W2080659660 abstract "The target of rapamycin (TOR) is a large (281 kDa) conserved Ser/Thr protein kinase that functions as a central controller of cell growth. TOR assembles into two distinct multiprotein complexes: TORC1 and TORC2. A defining feature of TORC1 is the interaction of TOR with KOG1 (Raptor in mammals) and its sensitivity to a rapamycin-FKBP12 complex. Here, we have reconstructed in three dimensions the 25 Å resolution structures of endogenous budding yeast TOR1 and a TOR-KOG1 complex, using electron microscopy. TOR features distinctive N-terminal HEAT repeats that form a curved tubular-shaped domain that associates with the C-terminal WD40 repeat domain of KOG1. The N terminus of KOG1 is in proximity to the TOR kinase domain, likely functioning to bring substrates into the vicinity of the catalytic region. A model is proposed for the molecular architecture of the TOR-KOG1 complex explaining its sensitivity to rapamycin. The target of rapamycin (TOR) is a large (281 kDa) conserved Ser/Thr protein kinase that functions as a central controller of cell growth. TOR assembles into two distinct multiprotein complexes: TORC1 and TORC2. A defining feature of TORC1 is the interaction of TOR with KOG1 (Raptor in mammals) and its sensitivity to a rapamycin-FKBP12 complex. Here, we have reconstructed in three dimensions the 25 Å resolution structures of endogenous budding yeast TOR1 and a TOR-KOG1 complex, using electron microscopy. TOR features distinctive N-terminal HEAT repeats that form a curved tubular-shaped domain that associates with the C-terminal WD40 repeat domain of KOG1. The N terminus of KOG1 is in proximity to the TOR kinase domain, likely functioning to bring substrates into the vicinity of the catalytic region. A model is proposed for the molecular architecture of the TOR-KOG1 complex explaining its sensitivity to rapamycin. Rapamycin is an antifungal agent used clinically as an immunosuppressant, with promising potential as an anticancer drug. On forming a complex with the immunophilin FKBP12, rapamycin suppresses cell growth by inhibiting the activity of the TOR protein (Inoki and Guan, 2006Inoki K. Guan K.L. Complexity of the TOR signaling network.Trends Cell Biol. 2006; 16: 206-212Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, Wullschleger et al., 2006Wullschleger S. Loewith R. Hall M.N. TOR signaling in growth and metabolism.Cell. 2006; 124: 471-484Abstract Full Text Full Text PDF PubMed Scopus (4367) Google Scholar). TOR is a member of the PI3-kinase-like protein kinases (PIKK), large molecular weight proteins (Inoki and Guan, 2006Inoki K. Guan K.L. Complexity of the TOR signaling network.Trends Cell Biol. 2006; 16: 206-212Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar) and functions as a central controller of cell growth by integrating signals from hormones, growth factors, and nutrients (Loewith et al., 2002Loewith R. Jacinto E. Wullschleger S. Lorberg A. Crespo J.L. Bonenfant D. Oppliger W. Jenoe P. Hall M.N. Two TOR complexes, only one of which is rapamycin sensitive, have distinct roles in cell growth control.Mol. Cell. 2002; 10: 457-468Abstract Full Text Full Text PDF PubMed Scopus (1360) Google Scholar, Wullschleger et al., 2006Wullschleger S. Loewith R. Hall M.N. TOR signaling in growth and metabolism.Cell. 2006; 124: 471-484Abstract Full Text Full Text PDF PubMed Scopus (4367) Google Scholar). Two genes, TOR1 and TOR2, were identified in yeast by means of genetic screens for mutations that rescued the antiproliferative effect of rapamycin (Heitman et al., 1991Heitman J. Movva N.R. Hall M.N. Targets for cell cycle arrest by the immunosuppressant rapamycin in yeast.Science. 1991; 253: 905-909Crossref PubMed Scopus (1417) Google Scholar). Yeast TOR1 and TOR2 share 67% sequence identity and are redundant in the rapamycin-sensitive signaling pathway (Loewith et al., 2002Loewith R. Jacinto E. Wullschleger S. Lorberg A. Crespo J.L. Bonenfant D. Oppliger W. Jenoe P. Hall M.N. Two TOR complexes, only one of which is rapamycin sensitive, have distinct roles in cell growth control.Mol. Cell. 2002; 10: 457-468Abstract Full Text Full Text PDF PubMed Scopus (1360) Google Scholar). Higher eukaryotes encode only a single TOR protein (mTOR in mammals), highly conserved with yeast TOR1 and TOR2 (40%–60% identity) (Guertin and Sabatini, 2005Guertin D.A. Sabatini D.M. An expanding role for mTOR in cancer.Trends Mol. Med. 2005; 11: 353-361Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar, Heitman et al., 1991Heitman J. Movva N.R. Hall M.N. Targets for cell cycle arrest by the immunosuppressant rapamycin in yeast.Science. 1991; 253: 905-909Crossref PubMed Scopus (1417) Google Scholar, Wullschleger et al., 2006Wullschleger S. Loewith R. Hall M.N. TOR signaling in growth and metabolism.Cell. 2006; 124: 471-484Abstract Full Text Full Text PDF PubMed Scopus (4367) Google Scholar). In vivo, TOR is associated with two distinct multiprotein complexes: TORC1 and TORC2 (Loewith et al., 2002Loewith R. Jacinto E. Wullschleger S. Lorberg A. Crespo J.L. Bonenfant D. Oppliger W. Jenoe P. Hall M.N. Two TOR complexes, only one of which is rapamycin sensitive, have distinct roles in cell growth control.Mol. Cell. 2002; 10: 457-468Abstract Full Text Full Text PDF PubMed Scopus (1360) Google Scholar). Yeast TORC1 comprises either TOR1 or TOR2 bound to KOG1, LST8, and Tco89, whereas TORC2 is formed from TOR2, AVO1, AVO1, AVO3, LST8, and Bit61 (Inoki and Guan, 2006Inoki K. Guan K.L. Complexity of the TOR signaling network.Trends Cell Biol. 2006; 16: 206-212Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, Loewith et al., 2002Loewith R. Jacinto E. Wullschleger S. Lorberg A. Crespo J.L. Bonenfant D. Oppliger W. Jenoe P. Hall M.N. Two TOR complexes, only one of which is rapamycin sensitive, have distinct roles in cell growth control.Mol. Cell. 2002; 10: 457-468Abstract Full Text Full Text PDF PubMed Scopus (1360) Google Scholar, Reinke et al., 2004Reinke A. Anderson S. McCaffery J.M. Yates 3rd, J. Aronova S. Chu S. Fairclough S. Iverson C. Wedaman K.P. Powers T. TOR complex 1 includes a novel component, Tco89p (YPL180w), and cooperates with Ssd1p to maintain cellular integrity in Saccharomyces cerevisiae.J. Biol. Chem. 2004; 279: 14752-14762Crossref PubMed Scopus (176) Google Scholar). KOG1 and AVO3 (Raptor and Rictor in mammals, respectively) confer distinctive structural and functional features on TORC1 and TORC2 (Hara et al., 2002Hara K. Maruki Y. Long X. Yoshino K. Oshiro N. Hidayat S. Tokunaga C. Avruch J. Yonezawa K. Raptor, a binding partner of target of rapamycin (TOR), mediates TOR action.Cell. 2002; 110: 177-189Abstract Full Text Full Text PDF PubMed Scopus (1365) Google Scholar, Inoki and Guan, 2006Inoki K. Guan K.L. Complexity of the TOR signaling network.Trends Cell Biol. 2006; 16: 206-212Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, Kim et al., 2002Kim D.H. Sarbassov D.D. Ali S.M. King J.E. Latek R.R. Erdjument-Bromage H. Tempst P. Sabatini D.M. mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery.Cell. 2002; 110: 163-175Abstract Full Text Full Text PDF PubMed Scopus (2189) Google Scholar, Kim et al., 2003Kim D.H. Sarbassov D.D. Ali S.M. Latek R.R. Guntur K.V. Erdjument-Bromage H. Tempst P. Sabatini D.M. GbetaL, a positive regulator of the rapamycin-sensitive pathway required for the nutrient-sensitive interaction between raptor and mTOR.Mol. Cell. 2003; 11: 895-904Abstract Full Text Full Text PDF PubMed Scopus (697) Google Scholar, Sarbassov et al., 2004Sarbassov D.D. Ali S.M. Kim D.H. Guertin D.A. Latek R.R. Erdjument-Bromage H. Tempst P. Sabatini D.M. Rictor, a novel binding partner of mTOR, defines a rapamycin-insensitive and raptor-independent pathway that regulates the cytoskeleton.Curr. Biol. 2004; 14: 1296-1302Abstract Full Text Full Text PDF PubMed Scopus (1984) Google Scholar, Wullschleger et al., 2005Wullschleger S. Loewith R. Oppliger W. Hall M.N. Molecular organization of target of rapamycin complex 2.J. Biol. Chem. 2005; 280: 30697-30704Crossref PubMed Scopus (177) Google Scholar). TORC1 is activated by growth factors, hormones, nutrients, and energy signals to regulate cell growth (Guertin and Sabatini, 2005Guertin D.A. Sabatini D.M. An expanding role for mTOR in cancer.Trends Mol. Med. 2005; 11: 353-361Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar, Wullschleger et al., 2006Wullschleger S. Loewith R. Hall M.N. TOR signaling in growth and metabolism.Cell. 2006; 124: 471-484Abstract Full Text Full Text PDF PubMed Scopus (4367) Google Scholar). Activated TORC1 phosphorylates two regulators of translation, S6K1 and 4E-BP1, thus stimulating protein synthesis. TORC1 is sensitive to inhibition by rapamycin through still obscure mechanisms. Rapamycin disrupts mTOR-Raptor interactions (Oshiro et al., 2004Oshiro N. Yoshino K. Hidayat S. Tokunaga C. Hara K. Eguchi S. Avruch J. Yonezawa K. Dissociation of raptor from mTOR is a mechanism of rapamycin-induced inhibition of mTOR function.Genes Cells. 2004; 9: 359-366Crossref PubMed Scopus (247) Google Scholar) but does not dissociate KOG1 from yeast TOR (Loewith et al., 2002Loewith R. Jacinto E. Wullschleger S. Lorberg A. Crespo J.L. Bonenfant D. Oppliger W. Jenoe P. Hall M.N. Two TOR complexes, only one of which is rapamycin sensitive, have distinct roles in cell growth control.Mol. Cell. 2002; 10: 457-468Abstract Full Text Full Text PDF PubMed Scopus (1360) Google Scholar). In contrast, TORC2 is resistant to rapamycin and regulates the actin cytoskeleton (Jacinto et al., 2004Jacinto E. Loewith R. Schmidt A. Lin S. Ruegg M.A. Hall A. Hall M.N. Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive.Nat. Cell Biol. 2004; 6: 1122-1128Crossref PubMed Scopus (1561) Google Scholar, Loewith et al., 2002Loewith R. Jacinto E. Wullschleger S. Lorberg A. Crespo J.L. Bonenfant D. Oppliger W. Jenoe P. Hall M.N. Two TOR complexes, only one of which is rapamycin sensitive, have distinct roles in cell growth control.Mol. Cell. 2002; 10: 457-468Abstract Full Text Full Text PDF PubMed Scopus (1360) Google Scholar). Structurally, TOR is defined by several conserved domains (Figure 1A and Figure S1, which is in the Supplemental Data available with this article online) (Wullschleger et al., 2006Wullschleger S. Loewith R. Hall M.N. TOR signaling in growth and metabolism.Cell. 2006; 124: 471-484Abstract Full Text Full Text PDF PubMed Scopus (4367) Google Scholar). The amino terminus is characterized by multiple huntingtin, elongation factor 3, a subunit of PP2A, and TOR1 (HEAT) repeats (Perry and Kleckner, 2003Perry J. Kleckner N. The ATRs, ATMs, and TORs are giant HEAT repeat proteins.Cell. 2003; 112: 151-155Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). Multiple adjacent HEAT motifs often adopt large extended superhelical structures (Groves and Barford, 1999Groves M.R. Barford D. Topological characteristics of helical repeat proteins.Curr. Opin. Struct. Biol. 1999; 9: 383-389Crossref PubMed Scopus (270) Google Scholar) and, specifically in mTOR, contribute to its association with Raptor (Kim et al., 2002Kim D.H. Sarbassov D.D. Ali S.M. King J.E. Latek R.R. Erdjument-Bromage H. Tempst P. Sabatini D.M. mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery.Cell. 2002; 110: 163-175Abstract Full Text Full Text PDF PubMed Scopus (2189) Google Scholar). The N-terminal HEAT repeat domain is followed by a C-terminal region conserved in PIKK proteins, comprising (1) an ∼600 amino acid helical FRAP/TOR, ATM, TRRAP (FAT) domain, also thought to be assembled from multiple HEAT repeats, (2) the catalytic kinase domain (∼370 residues), and (3) the short (∼70 residues) C-terminal FATC domain (Figure 1 and Figures S1 and S2). The carboxyl terminus also comprises the ∼100 amino acid FKBP12/FPR1-rapamycin binding (FRB) domain, which is immediately N-terminal to the kinase domain. The FRB domain consists of a pair of HEAT repeat units (Choi et al., 1996Choi J. Chen J. Schreiber S.L. Clardy J. Structure of the FKBP12-rapamycin complex interacting with the binding domain of human FRAP.Science. 1996; 273: 239-242Crossref PubMed Scopus (673) Google Scholar) and contains the serine residue (Ser1972 of yeast TOR1 and Ser1975 of yeast TOR2) that confers rapamycin resistance when substituted by arginine and isoleucine, respectively (Helliwell et al., 1994Helliwell S.B. Wagner P. Kunz J. Deuter-Reinhard M. Henriquez R. Hall M.N. TOR1 and TOR2 are structurally and functionally similar but not identical phosphatidylinositol kinase homologues in yeast.Mol. Biol. Cell. 1994; 5: 105-118Crossref PubMed Scopus (301) Google Scholar). Kontroller of growth 1 (KOG1) is a 176 kDa protein characterized by an amino-terminal Raptor N-terminal conserved (RNC) domain, four HEAT repeats, and seven C-terminal WD40 motifs (Loewith et al., 2002Loewith R. Jacinto E. Wullschleger S. Lorberg A. Crespo J.L. Bonenfant D. Oppliger W. Jenoe P. Hall M.N. Two TOR complexes, only one of which is rapamycin sensitive, have distinct roles in cell growth control.Mol. Cell. 2002; 10: 457-468Abstract Full Text Full Text PDF PubMed Scopus (1360) Google Scholar) (Figure 1B). KOG1 and its mammal homolog Raptor play a positive role in TOR signaling by functioning as a scaffold to recruit S6K1 and 4E-BP1 substrates to TORC1 (Kim et al., 2002Kim D.H. Sarbassov D.D. Ali S.M. King J.E. Latek R.R. Erdjument-Bromage H. Tempst P. Sabatini D.M. mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery.Cell. 2002; 110: 163-175Abstract Full Text Full Text PDF PubMed Scopus (2189) Google Scholar, Nojima et al., 2003Nojima H. Tokunaga C. Eguchi S. Oshiro N. Hidayat S. Yoshino K. Hara K. Tanaka N. Avruch J. Yonezawa K. The mammalian target of rapamycin (mTOR) partner, raptor, binds the mTOR substrates p70 S6 kinase and 4E–BP1 through their TOR signaling (TOS) motif.J. Biol. Chem. 2003; 278: 15461-15464Crossref PubMed Scopus (468) Google Scholar, Schalm et al., 2003Schalm S.S. Fingar D.C. Sabatini D.M. Blenis J. TOS motif-mediated raptor binding regulates 4E–BP1 multisite phosphorylation and function.Curr. Biol. 2003; 13: 797-806Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar, Wullschleger et al., 2006Wullschleger S. Loewith R. Hall M.N. TOR signaling in growth and metabolism.Cell. 2006; 124: 471-484Abstract Full Text Full Text PDF PubMed Scopus (4367) Google Scholar, Yonezawa et al., 2004Yonezawa K. Tokunaga C. Oshiro N. Yoshino K. Raptor, a binding partner of target of rapamycin.Biochem. Biophys. Res. Commun. 2004; 313: 437-441Crossref PubMed Scopus (64) Google Scholar). The TOS motifs of S6K1 and 4E-BP1 are recognized by the N-terminal RNC domain of KOG1/Raptor, greatly enhancing TOR-mediated substrate phosphorylation (Nojima et al., 2003Nojima H. Tokunaga C. Eguchi S. Oshiro N. Hidayat S. Yoshino K. Hara K. Tanaka N. Avruch J. Yonezawa K. The mammalian target of rapamycin (mTOR) partner, raptor, binds the mTOR substrates p70 S6 kinase and 4E–BP1 through their TOR signaling (TOS) motif.J. Biol. Chem. 2003; 278: 15461-15464Crossref PubMed Scopus (468) Google Scholar, Schalm et al., 2003Schalm S.S. Fingar D.C. Sabatini D.M. Blenis J. TOS motif-mediated raptor binding regulates 4E–BP1 multisite phosphorylation and function.Curr. Biol. 2003; 13: 797-806Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar). Additionally, KOG1/Raptor acts to stabilize TOR and confers the capacity to respond to upstream signals (Kim and Sabatini, 2004Kim D.H. Sabatini D.M. Raptor and mTOR: subunits of a nutrient-sensitive complex.Curr. Top. Microbiol. Immunol. 2004; 279: 259-270PubMed Google Scholar). Yeast lethal with sec thirteen (LST8) is a 34 kDa protein structurally defined by seven WD40 repeats. Mammalian LST8 binds to the kinase domain of mTOR and strongly stimulates the catalytic activity of TORC1 toward S6K1 and 4E-BP1 (Kim et al., 2003Kim D.H. Sarbassov D.D. Ali S.M. Latek R.R. Guntur K.V. Erdjument-Bromage H. Tempst P. Sabatini D.M. GbetaL, a positive regulator of the rapamycin-sensitive pathway required for the nutrient-sensitive interaction between raptor and mTOR.Mol. Cell. 2003; 11: 895-904Abstract Full Text Full Text PDF PubMed Scopus (697) Google Scholar, Wullschleger et al., 2006Wullschleger S. Loewith R. Hall M.N. TOR signaling in growth and metabolism.Cell. 2006; 124: 471-484Abstract Full Text Full Text PDF PubMed Scopus (4367) Google Scholar). The large size of TOR and its associated complexes, and the difficulties encountered in their isolation, have limited structural studies of these proteins. Here, we applied EM approaches to determine the structures of endogenously expressed yeast TOR1, and TORC1 containing TOR and KOG1. Our findings provide a three-dimensional view of TOR and explain how it interacts with KOG1, providing significant insights into TORC1 function. We used a tandem affinity purification (TAP) tag to purify proteins of the TORC1 complex. Yeast strains were generated with TOR1 tagged at its N terminus by means of homologous recombination of the TOR1 gene. Surprisingly, in the presence of low-detergent concentrations known to stabilize TORC1 (0.01% Tween 20) (Loewith et al., 2002Loewith R. Jacinto E. Wullschleger S. Lorberg A. Crespo J.L. Bonenfant D. Oppliger W. Jenoe P. Hall M.N. Two TOR complexes, only one of which is rapamycin sensitive, have distinct roles in cell growth control.Mol. Cell. 2002; 10: 457-468Abstract Full Text Full Text PDF PubMed Scopus (1360) Google Scholar), TOR1 eluted in the absence of the expected KOG1 and LST8 subunits (Figure 1Ca). However, this preparation provided homogenous TOR1, confirmed by MALDI-TOF mass spectrometry and similar in apparent molecular mass to that observed by Loewith et al., 2002Loewith R. Jacinto E. Wullschleger S. Lorberg A. Crespo J.L. Bonenfant D. Oppliger W. Jenoe P. Hall M.N. Two TOR complexes, only one of which is rapamycin sensitive, have distinct roles in cell growth control.Mol. Cell. 2002; 10: 457-468Abstract Full Text Full Text PDF PubMed Scopus (1360) Google Scholar, that was suitable for structural studies. To isolate TORC1 from yeast, KOG1 was tagged at its C terminus. KOG1 (identified by mass spectrometry) eluted from calmodulin Sepharose as a complex with TOR, as determined by mobility on SDS-PAGE relative to tagged TOR1 (Figure 1Cb). Potentially, the bulky TAP tag fused to the N terminus of TOR1 disrupted TOR1-KOG1 interactions. In the TORC1 preparation, the relative amounts of TOR and KOG1, as determined from silver staining on SDS gels, indicated a 1:1 complex. Furthermore, because only KOG1 was tagged, the eluted TOR would in principle consist of a mixture of TOR1 and TOR2. TOR1 and TOR2, which differ in mass by <0.2 kDa, cannot be distinguished by size on SDS-PAGE, and we were unable to obtain mass spectrometry data from a tryptic digest of the silver-stained band corresponding to TOR. However, relative to TOR1, the amount of TOR2 in TORC1 was reported to be small (Loewith et al., 2002Loewith R. Jacinto E. Wullschleger S. Lorberg A. Crespo J.L. Bonenfant D. Oppliger W. Jenoe P. Hall M.N. Two TOR complexes, only one of which is rapamycin sensitive, have distinct roles in cell growth control.Mol. Cell. 2002; 10: 457-468Abstract Full Text Full Text PDF PubMed Scopus (1360) Google Scholar). LST8 was not detected in any of our TOR1 and TORC1 preparations, either in the presence or absence of detergent, indicating that although LST8 may function to stabilize the mTOR-Raptor complex (Kim et al., 2003Kim D.H. Sarbassov D.D. Ali S.M. Latek R.R. Guntur K.V. Erdjument-Bromage H. Tempst P. Sabatini D.M. GbetaL, a positive regulator of the rapamycin-sensitive pathway required for the nutrient-sensitive interaction between raptor and mTOR.Mol. Cell. 2003; 11: 895-904Abstract Full Text Full Text PDF PubMed Scopus (697) Google Scholar), its presence is not essential for stabilizing core TORC1 from yeast. The purified TOR-KOG1 complex was concentrated to a level sufficient for visualization by using electron microscopy (Figure 1Cc). TOR1 degraded upon concentrating, and it was observed directly after elution. Because of the low quantities of TOR1 and TOR-KOG1 recovered after purification, we initially applied negative-stain electron microscopy (Figure 1D, only TOR1 shown, TOR-KOG1 not shown). The micrographs revealed that the majority of TOR1 presented a shape and dimensions similar to those observed previously for DNA-PKcs (Rivera-Calzada et al., 2005Rivera-Calzada A. Maman J.D. Spagnolo L. Pearl L.H. Llorca O. Three-dimensional structure and regulation of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs).Structure. 2005; 13: 243-255Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Previous data indicated that yeast TORC2 exists as both a monomer and a large oligomeric assembly (Wullschleger et al., 2005Wullschleger S. Loewith R. Oppliger W. Hall M.N. Molecular organization of target of rapamycin complex 2.J. Biol. Chem. 2005; 280: 30697-30704Crossref PubMed Scopus (177) Google Scholar). However, in most of our images, TOR1 and TORC1 were more consistent with a predominance of the monomeric form. Particles were also found corresponding to higher-order assemblies or aggregates, but these did not show an apparent structural order. Nevertheless, it is possible that our purification procedures could have disrupted ordered oligomers. Images of single molecules were extracted, and a total of 5643 particles for TOR1 and 5508 for TOR-KOG1 were subjected to iterative refinement (see Experimental Procedures) (Figures 1E and 1F). Importantly, our prior knowledge of the 3D structures of other PIKK proteins was not incorporated into this refinement. Purified TOR-KOG1 complex was also vitrified and observed under liquid nitrogen temperatures to attempt a higher-resolution cryo-EM structure determination (Figure 1G). The sample behaved inconsistently during the vitrification process that, combined with the very limited amount and concentration of the material recovered after purification and its tendency to degradation, resulted in the visualization of only a few-hundred TOR-KOG1 molecules. Two-dimensional averaging of the extracted particles indicated that the dimensions and general features of TORC1 visualized by cryo-EM were similar to those obtained with negative stain (Figure 1H). The 3D EM map of TOR1 at 25 Å resolution (Figures 2B–2E) revealed a molecule organized into a bulky and largely globular “head” domain connected to a flat tubular “arm” region, with overall dimensions of 140 × 115 × 80 Å. Such an arrangement of domains is closely reminiscent of the recently determined cryo-EM structure of DNA-PKcs (Rivera-Calzada et al., 2005Rivera-Calzada A. Maman J.D. Spagnolo L. Pearl L.H. Llorca O. Three-dimensional structure and regulation of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs).Structure. 2005; 13: 243-255Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar), with the main differences confined to the size and extent of the tubular regions (Figure 2A), consistent with the structural relationship of these two proteins (Figure S2). Significantly, the 3D structure of TOR1 could be superimposed onto the EM map of a DNA-PKcs/Ku70/Ku80 complex obtained with identical experimental conditions (microscope, magnification, and staining), revealing similarities in the organization of the head domain (Figure S3). We had previously annotated the cryo-EM structure of DNA-PKcs by using a combination of antibody labeling, bioinformatics, domain docking, and comparison with an EM structure of the related ATM (Rivera-Calzada et al., 2005Rivera-Calzada A. Maman J.D. Spagnolo L. Pearl L.H. Llorca O. Three-dimensional structure and regulation of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs).Structure. 2005; 13: 243-255Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar) (Figure S4). Aiding our interpretation of the TOR1 EM maps, we have now used the shared topology of DNA-PKcs and TOR1 to assign regions of the TOR1 3D map to conserved structural domains (Figures 2B–2E). Based on careful sequence alignments, Perry and Kleckner, 2003Perry J. Kleckner N. The ATRs, ATMs, and TORs are giant HEAT repeat proteins.Cell. 2003; 112: 151-155Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar proposed that PIKK proteins are giant helical HEAT repeat molecules, such that the entire region N-terminal to the PIKK catalytic domain constitutes HEAT repeats (∼2000 residues, some 50 HEAT repeats). This notion is consistent with our analysis using fold-recognition programs (D.B., unpublished data) that predict strong similarity to HEAT/ARM repeats of the protein phosphatase PR65/A subunit and β-importin for residues 1–2025 of TOR1 (Figure S1). A prominent shared feature of the 3D structures of DNA-PKcs and TOR1 is a curved tubular-shaped extended arm-like domain (Figure 2, orange). In DNA-PKcs, this was assigned as a portion of its N-terminal HEAT repeats (Rivera-Calzada et al., 2005Rivera-Calzada A. Maman J.D. Spagnolo L. Pearl L.H. Llorca O. Three-dimensional structure and regulation of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs).Structure. 2005; 13: 243-255Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar) (Figure S4), suggesting that the equivalent structure of TOR1 also corresponds to its more N-terminal HEAT repeats. Consistent with such an assignment, the size and overall dimensions of this domain vary among DNA-PKcs, ATM (Rivera-Calzada et al., 2005Rivera-Calzada A. Maman J.D. Spagnolo L. Pearl L.H. Llorca O. Three-dimensional structure and regulation of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs).Structure. 2005; 13: 243-255Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar), and TOR proteins, whereas the head domain remains fairly constant. Compared with TOR, DNA-PKcs comprises an additional 1500 residues, likely to represent ∼35 HEAT repeats (Perry and Kleckner, 2003Perry J. Kleckner N. The ATRs, ATMs, and TORs are giant HEAT repeat proteins.Cell. 2003; 112: 151-155Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar), which are assumed to be accommodated within the greatly extended arm-like structure of DNA-PKcs relative to TOR (Figure 2 and Figure S2). The arm-like domain of TOR1 (Figure 2, orange) was estimated to represent two-thirds of its N-terminal HEAT repeats (excluding the FAT and helical domains, Figure S1) with the remaining HEAT motifs adopting a more globular structure adjacent to the arm domain (Figure 2, yellow). In the crystal structure of PI3Kγ, an N-terminal ∼200 residue five-unit HEAT repeat structure (helical domain) forms an extensive interface with the lipid kinase catalytic domain, generating a contiguous globular core, collectively termed the helical/kinase domain (Walker et al., 1999Walker E.H. Perisic O. Ried C. Stephens L. Williams R.L. Structural insights into phosphoinositide 3-kinase catalysis and signalling.Nature. 1999; 402: 313-320Crossref PubMed Scopus (396) Google Scholar). Such a helical region would correspond to residues 1802–2026 of TOR1 (Figure 1A and Figure S1) and incorporates both the N-terminal region of FRB (the remainder of which is formed from the N-terminal helices of the kinase domain) and the C-terminal region of the FAT domain. The reconstruction of TOR1 features a head region (Figures 2B–2E, yellow, pink, and green) with a more globular domain at its center (Figure 2, pink) that in DNA-PKcs was unambiguously fitted to the atomic structure of the helical/kinase domain of PI3Kγ (Walker et al., 1999Walker E.H. Perisic O. Ried C. Stephens L. Williams R.L. Structural insights into phosphoinositide 3-kinase catalysis and signalling.Nature. 1999; 402: 313-320Crossref PubMed Scopus (396) Google Scholar) (Figure S4). By analogy to DNA-PKcs, this region in TOR1 (Figure 2, pink) would correspond to the helical/catalytic domain, incorporating the FRB domain (Figure 2D, proposed location of FRB labeled with an asterisk). The FATC domain forms an extended helical region (Dames et al., 2005Dames S.A. Mulet J.M. Rathgeb-Szabo K. Hall M.N. Grzesiek S. The solution structure of the FATC domain of the protein kinase target of rapamycin suggests a role for redox-dependent structural and cellular stability.J. Biol. Chem. 2005; 280: 20558-20564Crossref PubMed Scopus (107) Google Scholar) attached to the catalytic domain C terminus (Figure 2, blue). Importantly, the FATC domain is apparently flattened on negative staining of DNA-PKcs (Rivera-Calzada et al., 2005Rivera-Calzada A. Maman J.D. Spagnolo L. Pearl L.H. Llorca O. Three-dimensional structure and regulation of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs).Structure. 2005; 13: 243-255Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar) and TOR1. The HEAT repeats corresponding to the FAT domain (some 600 residues) (Figure 1A and Figure S1) cannot be precisely mapped within the EM electron density. However, since the C-terminal 100 residues of the FAT domain correspond to the N-terminal 100 residues of the helical region of the helical/kinase domain, it is reasonable to assume the FAT domain will be in close proximity to, and extend structurally, the helical region of the helical/kinase domain, contributing toward the remaining unassigned regions of the EM map (Figure 2, green) as proposed for DNA-PKcs (Rivera-Calzada et al., 2005Rivera-Calzada A. Maman J.D. Spagnolo L. Pearl L.H. Llorca O. Three-dimensional structure and regulation of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs).Structure. 2005; 13: 243-255Abstract Full Text Full Text PDF PubMed Scopus (90) Google" @default.
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