Architecture of the human mTORC2 core complex
The serine/threonine kinase mammalian target of rapamycin ( mTOR ) is the master regulator of cellular growth ( Saxton and Sabatini , 2017 ) . Originally identified in yeast as TOR ( Heitman et al . , 1991; Kunz et al . , 1993 ) , the mammalian orthologue mTOR senses a range of cellular cues , including nutrient and energy availability , and growth factor signals , to control metabolism and autophagy ( Laplante and Sabatini , 2012; Loewith and Hall , 2011; Saxton and Sabatini , 2017 ) . Due to its role as a central controller of cell growth , aberrant mTOR signaling is observed in major diseases ( Dazert and Hall , 2011; Saxton and Sabatini , 2017 ) . mTOR is a member of the phosphatidylinositol-kinase-related kinase ( PIKK ) family ( Keith and Schreiber , 1995 ) and exerts its function as the enzymatic component of two distinct protein complexes , mTOR complex ( mTORC ) one and mTORC2 . Together with the small protein mLST8 , mTOR forms the core of both complexes ( Kim et al . , 2003; Loewith et al . , 2002 ) . The protein Raptor is unique to mTORC1 ( Hara et al . , 2002; Kim et al . , 2002 ) , whereas the proteins Rictor and SIN1 are defining subunits of mTORC2 ( Frias et al . , 2006; Jacinto et al . , 2006; Jacinto et al . , 2004; Sarbassov et al . , 2004; Yang et al . , 2006 ) . Additionally , Protor-1/2 and Deptor can bind to mTORC2 ( Pearce et al . , 2007; Peterson et al . , 2009 ) . The polyketide rapamycin , in complex with the endogenous protein FKBP12 , inhibits mTORC1 activity by binding the FKBP-rapamycin ( FRB ) domain of mTOR ( Aylett et al . , 2016; Yang et al . , 2013 ) . In contrast , the FKBP-rapamycin complex is unable to bind mTORC2 ( Jacinto et al . , 2004 ) , although prolonged rapamycin treatment disrupts mTORC2 signaling by preventing mTOR incorporation into mTORC2 ( Lamming et al . , 2012; Sarbassov et al . , 2006 ) . mTORC2 is activated by growth factors , in particular through the insulin/PI3K signaling pathway ( Saxton and Sabatini , 2017 ) . The major substrates of mTORC2 are the AGC-kinase family members Akt , SGK-1 and PKC-α ( García-Martínez and Alessi , 2008; Ikenoue et al . , 2008; Sarbassov et al . , 2005 ) . Biochemical analysis has revealed that mTORCs form dimeric assemblies ( Wullschleger et al . , 2005 ) . The architecture of mTORC1 was resolved only recently through the use of cryo-EM and X-ray crystallography ( Aylett et al . , 2016; Yang et al . , 2016 ) . However , although biochemical analysis and a 26 Å EM reconstruction of yeast TORC2 have suggested that TOR and Lst8 form the same dimeric core as observed in mTORC1 ( Gaubitz et al . , 2015; Wullschleger et al . , 2005 ) , higher resolution information on the mTORC2 assembly remains unavailable . Structural data on mTORC2 accessory proteins are restricted to the fission yeast Sin1 CRIM domain ( Tatebe et al . , 2017 ) and the SIN1 PH domains ( Pan and Matsuura , 2012 ) .
We co-expressed the core components of human mTORC2; mTOR , mLST8 , Rictor , SIN1 and Protor-1 , in Spodoptera frugiperda cells using the MultiBac system ( Fitzgerald et al . , 2006 ) . Expression was attained for all components , and mTORC2 was captured by affinity to a FLAG tag inserted in the amino-terminal HEAT repeats of mTOR . Reconstituted complexes were purified by size-exclusion chromatography ( SEC ) . Yields remained low ( ~0 . 1 nmol complex per 10 L culture ) , as mTORC2 proved labile over the course of purification , resulting in two major species of mTOR complexes eluting from SEC; mTOR-mLST8 alone and reconstituted mTORC2 complex comprising mTOR , mLST8 , Rictor , SIN1 and substoichiometric amounts of Protor-1 , as confirmed by mass spectrometry ( Figure 1—figure supplement 1 ) . Human mTORC2 demonstrated kinase activity towards the substrate Akt-1 ( Figure 1—figure supplement 1 ) . mTORC2 also proved extremely labile during sample preparation for both negative staining ( Figure 1—figure supplement 2 ) and cryo-electron microscopy ( cryo-EM ) . Stabilisation of human mTORC2 , by cross-linking with glutaraldehyde using a modified gradient fixation protocol , allowed us to visualise intact , rhombic complexes . We collected cryo-EM data from stabilised samples , yielding 94 953 mTOR-dimer-containing particles for single particle analysis ( Figure 1—figure supplement 3 ) . Due to the labile nature of the complex , we observed intact complexes only in thicker ice , which limited the achievable contrast . Single particle reconstruction of mTORC2 proceeded from both an ab initio model generated from negatively stained samples and from the prior structure of the Kluyveromyces marxianus TOR-Lst8 complex ( Baretić et al . , 2016 ) , yielding essentially identical results . A reconstruction of non-cross-linked mTORC2 from negatively stained grids agrees well with the cryo-EM reconstructions of cross-linked mTORC2 indicating that the cross-linking procedure preserves the native structure of the complex ( Figure 1—figure supplement 2 ) . mTORC2 proved heterogeneous; refinement initiated from the K . m . TOR-Lst8 reference yielded classes encompassing free mTOR-mLST8 ( 30% ) , mTOR-mLST8 occupied on a single flank by accessory protein ( 52% ) and a complete complex bearing accessory factors on both flanks of the mTOR dimer ( 18% ) ( Figure 1—figure supplement 4 ) . Since only a fraction of particles were of the complete complex we conclude that our mTORC2 sample dissociated prior to fixation or due to non-exhaustive cross-linking . Subsequent refinement of the complete complex with the application of C2 symmetry resolved the entire complex to 7 . 4 Å , while a focused refinement on the accessory factor density yielded 6 . 2 Å resolution , based upon a gold standard FSC of 0 . 143 ( Figure 1—figure supplement 3 ) . The resolution is likely limited by the achieved contrast in the measured micrographs and the inherent flexibility of the Rictor/SIN1 domains . The complete human mTORC2 dimer displays a general organization similar to that we previously reported for mTORC1 ( Aylett et al . , 2016 ) ( Figure 1 ) . The previous 26 Å structure of Saccharomyces cerevisiae TORC2 was refined without symmetry because of substantial differences in the densities resolved on either side of the central cavity ( Gaubitz et al . , 2015 ) ( Figure 1—figure supplement 5 ) , however , our complete mTORC2 complex is effectively identical ( real-space two-fold rotated correlation >0 . 9 ) on either side of the symmetry axis to at least 8 . 9 Å ( the limit of resolution in C1 ) refined without symmetry ( Figure 1 and Figure 1—figure supplement 3 ) . Both mTORC1 and mTORC2 therefore function as dimeric , C2 symmetric , complexes . The dimeric arrangement of mTOR in mTORC2 is most similar to that observed for the K . m . TOR-Lst8 ( Baretić et al . , 2016 ) ( Figure 1—figure supplement 5 ) . The FAT domains of the ( m ) TOR dimer in mTORC2 and TOR-Lst8 come within 6 Å of each other , contrary to the conformation of the mTOR dimer in mTORC1 that is substantially outward-rotated . The resemblance between the free mTOR dimer and the mTOR dimer found within the mTORC2 complex may account for the increased lability of mTORC2 compared to mTORC1 . Rictor makes up most ( ~60% ) of the mass of the mTOR accessory proteins and is predicted to have an almost entirely helical secondary structure , most of which is expected to be α-solenoidal . SIN1 consists of two domains of known structure , a CRIM domain and a PH domain , neither of which form α-solenoids . While it remains impossible to definitively trace a polypeptide chain at this resolution , we were able to identify five regions of stacked α-solenoidal repeats , of which three can be resolved to the level of individual helices and two are less well ordered , that make up the majority of the density outside of the mTOR-mLST8 dimer and account for most of the elements expected to correspond to Rictor ( Figure 2A ) . The assignment of the major density features to Rictor is confirmed by a negatively stained EM reconstruction of mTORC2 lacking Protor-1 , which recapitulates density for all but one partially disordered extremus of the accessory protein region ( Figure 2—figure supplement 1 ) . The most striking feature , which we dub the ‘tower’ , is a well-resolved , 9/10-α-hairpin stack rising from the ‘bridge’ HEAT repeat of mTOR . This centrepiece is joined at one-third its length from the bridge by another well-resolved α-solenoid , encompassing 4/5-hairpins , which we refer to as the ‘buttress’ . The buttress links the tower to a region we term the ‘body’ , comprising three further repeats ( 9–11 hairpins in total ) that stack against one another , with its periphery being poorly ordered . The body is only peripherally associated with the mTOR dimer . Finally , at the top of the tower , a ‘cap’ region of linked density juts out toward the mTOR active site . No large regions of non-α-solenoidal density were resolved , preventing us from assigning a location to SIN1 , although a small region of non-helical density at the extremity of the cap represents the most likely candidate . This location of SIN1 would corroborate previous studies implying an interaction between SIN1 and mLST8 and a role of the SIN1 CRIM domain in recruiting substrates towards the active site of mTOR within mTORC2 ( Tatebe et al . , 2017 ) . The principal site of interaction between Rictor and the mTOR-mLST8 dimer is the juncture of the bridge and the horn , which is occupied by the base of the tower and its contact with the buttress . This is the same site occupied by the characteristic mTORC1 subunit Raptor ( Figure 2C ) . Baretic and colleagues have established that the TOR dimer interface runs along this plane , and have shown that a partner protein is not required for TOR dimer formation ( Baretić et al . , 2016 ) . We confirm this observation for mTOR-mLST8 , demonstrating that the Rictor-mTOR and Raptor-mTOR interactions can be formed only with intact mTOR dimer and are not required to stabilise the mTOR dimer . Given that the inter-subunit interfaces are completely different in the two mTOR complexes ( in Raptor the cleft between the horn and bridge is occupied by the loops at the tips of three helical hairpins , whereas in mTORC2 a single helix covers the length of the cleft ) , there must be an as yet unknown selective advantage to a dimeric architecture . Another less well-resolved contact is the junction of the body and the free end of the bridge amino-terminal HEAT repeats within the mTOR dimer , which does not recapitulate any contact found in mTORC1 . Regions of Raptor make contact with the HEAT repeat horn in the mTORC1 structure , however , the horn does not appear to make an analogous contact with any mTORC2 accessory protein . The differences between mTORC1 and mTORC2 in the mTOR-exposed surfaces of the horn region are presumably linked to their distinct modes of regulation by post-translational modifications or binding of associated protein factors . The final major contact identified between Rictor and the mTOR dimer is near the FRB domain of mTOR . The tower α-solenoid repeat bridges the gap between the binding site at the juncture of the mTOR dimer and the FRB domain of mTOR itself , while the cap , which is connected to the tower , straddles the surface of the FRB , deepening the active site cleft of the mTOR kinase domain . Raptor similarly deepens the active site in mTORC1 , but from a position directly opposite mLST8 , as opposed to Rictor , which approaches from the other side of the active site . This position of the cap masks the FKBP-rapamycin binding site of mTOR , thereby explaining the rapamycin insensitivity of mTORC2 ( Figure 2B ) . This finding is consistent with what was seen previously with yeast TORC2 ( Gaubitz et al . , 2015 ) . It is tempting to conclude that this region is responsible for substrate recruitment , given its proximity to the active site , however , further study is required to confirm this . The architecture of human mTORC2 reveals that the two mTOR complexes share many features , including the preservation of effective C2 symmetry , the common binding site for the principal accessory proteins Raptor and Rictor , the deepening of the active site cleft and the significant distance between the presumptive substrate-selection region and the active site of mTOR . While this manuscript was under revision , another publication described the architecture of yeast TORC2 at a resolution of 7 . 9 Å ( Karuppasamy et al . , 2017 ) . The main conclusions drawn in that publication are consistent with our findings . Many important biological questions remain , in particular the reason for the requirement for C2 symmetry , and the exact identity and function of the mTORC2 domains making up the cap region . A full interpretation of human mTORC2 must await future higher-resolution studies of a more stable form of the complex .
Unless otherwise stipulated , Sf21 insect cell lines and media were provided by Expression Systems ( Davies , USA ) and Lonza ( Basel , Switzerland ) , respectively . Cell lines have been tested negative for mycoplasma contamination using the MycoAlert mycoplasma detection kit from Lonza ( Basel , Switzerland ) . Chromatography equipment was provided by GE Healthcare ( Schenectady , USA ) , chemicals were provided by Sigma Aldrich ( St . Louis , USA ) and Applichem ( Darmstadt , Germany ) , electron microscopy consumables were provided by Agar Scientific ( Stansted , UK ) , and molecular graphics were generated using Chimera ( Pettersen et al . , 2004 ) , Coot ( Emsley et al . , 2010 ) and PyMol ( Schrödinger LLC , New York , USA ) . Expression plasmids were prepared by cloning an internal FLAG-tag into pAB2G-mTOR after Asp258 , the coding sequence of Rictor into pIDK , SIN1 into pAB1G and mLST8 into pIDC . A triple pIDC expression plasmid encoding mLST8 , SIN1 and Protor-1 was synthesized de novo ( Genscript , Piscataway , USA ) . Rictor was originally amplified from myc-Rictor corrected , which was a gift from David Sabatini ( Sarbassov et al . , 2004 ) ( Addgene plasmid #11367 ) . In order to clone tandem-tomato ( tdTomato ) labeled SIN1 , the tdTomato cDNA was amplified from tdTomato-N1 and inserted into pAB1G-SIN1 using the In-Fusion HD cloning kit ( Takara Bio USA , Mountain View , USA ) . tdTomato-N1 was kindly provided by M . Davidson and R . Tsien ( Addgene plasmid #54642 ) . The complex of H . s . mTORC2 was expressed in Sf21 cells using the ‘MultiBac’ Baculovirus expression system ( Fitzgerald et al . , 2006 ) ( Geneva Biotech , Geneva , Switzerland ) . Briefly , expression plasmids encoding FLAG-tagged mTOR , Rictor and the triple expression plasmid encoding SIN1 , mLST8 , and Protor-1 were fused to a ‘MultiBac’ expression plasmid using Cre-recombinase ( New England Biolabs , Ipswich , USA ) and recombined with a bacmid for baculovirus production . In order to express mTORC2-ΔProtor-1 the plasmids encoding Flag-tagged mTOR , Rictor and mLST8 were fused to a ‘MultiBac’ expression plasmid and recombined with a bacmid for baculovirus production . Baculovirus for the expression of tdTomato tagged SIN1 was produced separately . For the expression and purification of mTORC2-ΔProtor-1 , Sf21 cells were coinfected with baculovirus encoding Flag-mTOR , Rictor and mLST8 and baculovirus encoding SIN1-tdTomato . Sf21 cells expressing mTORC2 or mTORC2-ΔProtor-1 were harvested 72 hr post-infection and lysed in 50 mM bicine pH 8 . 5 , 200 mM NaCl , 2 mM MgCl2 lysis buffer by sonication . The lysate was cleared by centrifugation at 234 788 · g and then incubated with anti- DYKDDDDK agarose beads ( Genscript , Piscataway , USA ) for 2 hr at 4°C . The beads were washed 4 times in 50 mM bicine pH 8 . 5 , 200 mM NaCl and bound protein was eluted with DYKDDDDK-peptide ( 0 . 6 mg/ml; Genscript , Piscataway , USA ) . The eluted protein was concentrated to 0 . 5 ml and applied to a tandem Superose 6 Increase 10/300 GL gel filtration column equilibrated in 10 mM bicine pH8 . 5 , 150 mM NaCl , 0 . 5 mM EDTA , 2 mM TCEP . Fractions corresponding to mTORC2 were pooled , concentrated and directly used for further experiments . mTORC2 kinase activity assays were conducted in 25 mM HEPES pH 7 . 4 , 100 mM potassium acetate , 2 mM MgCl2 using Akt-1 ( Jena Bioscience , Jena , Germany ) as a substrate . The indicated gel filtration fractions of a mTORC2 preparation were pooled and concentrated . In a 50 µl reaction volume 8 . 5 µg mTORC2 or mTOR/mLST8 were mixed with 100 ng inactive Akt-1 in reaction buffer and where indicated with 20 µM Torin1 . The mixture was pre-incubated for 5 min on ice , and the reaction was initiated with the addition of 50 µM ATP . After 20 min at 37°C the reaction was terminated by the addition of 12 . 5 µl 5x SDS loading buffer . The reactions were Western Blotted using primary antibodies against phospho-Akt-Ser473 ( #4060 ) , Akt ( #4685 ) , and mTOR ( #2972; Cell Signaling Technologies , Beverly , USA ) at a dilution of 1:1000 . A goat anti-rabbit HRP-labeled antibody ( ab6721; Abcam , Cambridge , UK ) was used as the secondary antibody at a dilution of 1:3000 . Given that H . s . mTORC2 proved highly labile , it was purified , fixed and quenched using a modification of the GraFix protocol ( Kastner et al . , 2008 ) . Briefly , 7 . 5–27 . 5% ( w/v ) gradients were prepared from 150 mM NaCl , 15 mM NaBicine pH 8 . 0 , 1 mM TCEP . The upper quarter of the less dense buffer was supplemented with 0 . 25% glutaraldehyde ( Grade I ) , whereas the lower quarter of the denser buffer was supplemented with 150 mM NH4Cl . Samples were subjected to ultracentrifugation for 16 hr at 100 000 ∙ g in an SW32 rotor , thereby both fixing and quenching the sample in situ , and recovered by gradient fractionation . The recovered peak was concentrated to as low a volume as possible in 100 kDa cutoff Vivaspin concentrators ( Sartorius Stedim Biotech , Aubagne , France ) . The buffer was then exchanged three times against 150 mM NaCl , 15 mM NaBicine pH 8 . 0 , 1 mM TCEP in order to minimise the residual sugar in the final sample . A fixed sample of mTORC2 was applied to a carbon coated holey carbon grid and stained with 2% ( w/v ) uranyl acetate . Micrographs ( 33 ) were collected using an FEI F20 electron microscope ( Thermo Fisher Scientific , Waltham , MA , USA ) at a magnification of 82 000 fold , an acceleration voltage of 200 kV , and a total dose of 20 e/A2 at a defocus of between −0 . 5 and −2 . 0 μm . Particles ( 15 331 ) were selected semi-automatically using BOXER ( Ludtke et al . , 1999 ) . The parameters of the contrast transfer function were then determined with CTFFIND4 ( Rohou and Grigorieff , 2015 ) . Particles were 2D-classified into 100 classes in two dimensions using RELION ( Scheres , 2012 ) and sixteen well-defined classes were selected for initial three-dimensional reconstruction . Initial models were created using the e2initialmodel . py function in EMAN 2 . 1 ( Tang et al . , 2007 ) , then filtered to 60 Å and used as an initial reference for gold-standard refinement . The resulting initial model ( resolution 26 Å , gold-standard FSC ) was used for further refinement . We prepared EM samples from the peak fraction of the final Superose 6 Increase gel filtration step by negative staining . The sample was adsorbed to a freshly glow discharged thin carbon film supported on a 200 mesh copper grid and stained with 2% ( w/v ) uranyl acetate solution for 1 min with one additional post-staining step . The grid was imaged on a Philips CM 100 operated at 13700 fold nominal magnification at 80 kV acceleration voltage with an Olympus Veleta camera resulting in a pixel size of 5 . 4 Å on the specimen level . Images were acquired at a defocus between −0 . 9 and −1 . 7 μm . Particles ( 13879 ) were selected semi-automatically using BOXER ( Ludtke et al . , 1999 ) . Contrast transfer function parameters were determined with CTFFIND4 ( Rohou and Grigorieff , 2015 ) . All further processing steps were done within the RELION software ( Scheres , 2012 ) . Initially , particles were classified into 50 classes followed by a second round of classification into 80 classes ( Figure 1—figure supplement 2 ) . Well-defined average images were selected , and the subset of particles which contributed to these average images was chosen for further processing . An initial model was created without applying symmetry using the 3D initial model function of RELION , which utilizes a stochastic gradient descent algorithm for de novo structure determination . The initial model was refined and used in a 3D classification into three classes applying C2 symmetry . One class ( 1261 particles ) showed well-defined density for the core and the peripheral domains ( Figure 1—figure supplement 2 ) . This reconstruction ( 29 Å resolution ) was superimposed on the cryo-EM reconstruction using UCSF CHIMERA ( Pettersen et al . , 2004 ) . We prepared EM samples from affinity-purified mTORC2-ΔProtor-1 by crosslinking and negative staining . Briefly , a sample of the mTORC2-ΔProtor-1 complex was stabilised by fixation with 0 . 1% ( v/v ) glutaraldehyde for 15 min on ice . The sample was adsorbed to a thin carbon film and stained with 2% ( w/v ) uranyl acetate solution for 2 min with three additional post-staining steps . The sample was imaged on a Tecnai F20 electron microscope operated at 82 000 fold magnification at 200 kV acceleration voltage . Images were acquired under low dose conditions with a total dose of 20 e/Å2 at −1 to −2 μm defocus on a Gatan US 4 000 CCD camera . A total of 35 micrographs were selected for further processing in Relion2 ( Scheres , 2012 ) using contrast transfer function correction with CTFFIND4 ( Rohou and Grigorieff , 2015 ) . Single particle images were picked with Relion using a gaussian picking reference . After two-dimensional classification 13 170 single particle images were selected for refinement , during which C2 symmetry was applied . The structure of the mTORC2-ΔProtor-1 complex was refined to a final resolution of 21 Å ( gold-standard FSC , 0 . 143 criterion ) ( Scheres , 2012 ) . Samples of mTORC2 were applied to holey carbon copper grids ( R2/2 – Quantifoil ) bearing an additional fine film of carbon . Grids were blotted for two seconds and then plunged directly into a mixture of liquid ethane ( 33% ) and propane ( 67% ) using a vitrobot mark 4 ( Thermo Fisher Scientific , Waltham , MA , USA ) at 4°C and 95% humidity . Data were recorded semi-automatically using SerialEM ( Mastronarde , 2005 ) on a Titan Krios transmission electron microscope ( Thermo Fisher Scientific , Waltham , MA , USA ) equipped with a K2 Summit direct electron detector ( GATAN , San Diego , USA ) at 300 kV , 47 100 fold magnification and with an applied defocus of between −1 . 0 and −3 . 0 μm , resulting in 3 997 images of 3 838 by 3 710 pixels with a pixel size of 1 . 06 Å on the object scale . Each image was recorded as forty separate frames in electron counting mode , comprising a total exposure of 80 e-Å−2 , which were subsequently aligned , summed and weighted by dose according to the method of Grant and Grigorieff ( Grant and Grigorieff , 2015 ) using Motioncor2 ( Zheng et al . , 2017 ) to obtain the final image . Poor quality micrographs were rejected based on the diminished maximum resolution and regularity of the Thon rings observed in the power spectra . Estimation of the contrast transfer function was carried out for each image using CTFFIND4 ( Rohou and Grigorieff , 2015 ) , particles were selected using BATCHBOXER ( Ludtke et al . , 1999 ) , and refinement thereafter performed using RELION ( Scheres , 2012 ) . Two cycles of two-dimensional reference-free alignment of 207 920 boxed particles into 100 classes were performed initially , and particles that did not yield high-resolution class averages were excluded from further refinement . Three-dimensional classification of the remaining 94 953 mTORC2 particles into six classes was then performed with RELION ( Scheres , 2012 ) , separating the dataset into classes without excess density ( mTOR-mLST8 - 28 774 ) , occupied on one flank ( C1-mTORC2 - 48 996 ) , and doubly occupied ( C2-mTORC2 - 17 183 ) . The C2 particles were refined independently ( gold-standard ) using full-sized images , resulting in a complete symmetrical mTORC2 density map with an estimated resolution of 7 . 4 Å at a Fourier shell correlation of 0 . 143 ( Scheres , 2012 ) . Resolution was limited by the poor cryo-stability of the complex , the necessity for heavy fixation of the sample , and by conformational flexibility . Additionally , the mTOR-mLST8 core dominated the overall particle alignment , resulting in lower local resolution for the accessory proteins due to their flexibility . In order to attain the highest possible resolution of the accessory factors , the C1-mTORC2 particles and the two symmetrical sides of each C2-mTORC2 particle were refined in combination by subtracting the symmetry-related density and refining all occupied sides of the molecule in a single class , yielding a focused accessory factor density map with an estimated resolution of 6 . 2 Å at a Fourier shell correlation of 0 . 143 ( Scheres , 2012 ) . The cryo-EM density map representing the complete structure of mTORC2 with C2 symmetry has been deposited in the EM Databank under accession code EMDB ID EMD-3927 . The density corresponding to the focused refinement on the accessory proteins of mTORC2 has been deposited in the EM Databank under accession code EMDB ID EMD-3928 .
The mammalian target of rapamycin ( mTOR ) is a key protein kinase controlling cellular metabolism and growth . It is part of the two structurally and functionally distinct multiprotein complexes mTORC1 and mTORC2 . Dysregulation of mTOR occurs in diabetes , cancer and neurological disease . We report the architecture of human mTORC2 at intermediate resolution , revealing a conserved binding site for accessory proteins on mTOR and explaining the structural basis for the rapamycin insensitivity of the complex .
To grow and multiply , a living cell must take a variety of factors into account , such as its own energy levels and the availability of nutrients . A protein called mTOR sits at the core of a signaling pathway that integrates these and other sources information . Problems with the mTOR pathway contribute to several diseases including diabetes and cancer . The mTOR protein occurs in two distinct protein complexes , called mTORC1 and mTORC2 . These complexes contain a mix of other proteins – known as accessory proteins . They also sense different cues and act upon distinct targets in the cell . Recent research reported the structure of mTORC1 , which provided clues about how this complex works . Yet , much less was known about the mTORC2 complex . Stuttfeld , Aylett et al . have now used a technique called cryo-electron microscopy to reveal the three-dimensional architecture of the human version of mTORC2 . Comparing the new mTORC2 structure to the existing one for mTORC1 showed that they have many features in common but important differences too . The overall shape of both complexes is similar and each complex contains two copies of mTOR arranged in a similar way . Also , the main accessory proteins in each complex interact with almost the exact same parts of mTOR , but the accessory proteins in mTORC2 are organized differently from those of mTORC1 . The different accessory proteins also have distinct shapes . These differences could help to explain why the complexes respond to different cues and recognize different targets . These new findings provide an entry point for further studies on how mTORC2 works in cells . The next step is to get a higher resolution image of the structure of this complex to see the finer details of all the components . This may in the future help scientists to develop drugs that inhibit mTORC2 to treat cancer and other diseases .