RESEARCH ARTICLE◥

STRUCTURAL BIOLOGY

Architecture of human Rag GTPase heterodimersand their complex with mTORC1Madhanagopal Anandapadamanaban1, Glenn R. Masson1, Olga Perisic1, Alex Berndt1*,Jonathan Kaufman1, Chris M. Johnson1, Balaji Santhanam1, Kacper B. Rogala2,David M. Sabatini2,3,4,5,6, Roger L. Williams1†

The Rag guanosine triphosphatases (GTPases) recruit the master kinase mTORC1 to lysosomes toregulate cell growth and proliferation in response to amino acid availability. The nucleotide state of Ragheterodimers is critical for their association with mTORC1. Our cryo–electron microscopy structure ofRagA/RagC in complex with mTORC1 shows the details of RagA/RagC binding to the RAPTOR subunitof mTORC1 and explains why only the RagAGTP/RagCGDP nucleotide state binds mTORC1. Previous kineticstudies suggested that GTP binding to one Rag locks the heterodimer to prevent GTP binding to theother. Our crystal structures and dynamics of RagA/RagC show themechanism for this locking and explain howoncogenic hotspotmutations disrupt this process. In contrast to allosteric activation by RHEB, Rag heterodimerbinding does not change mTORC1 conformation and activates mTORC1 by targeting it to lysosomes.

Themechanistic target of rapamycin com-plex 1 (mTORC1) is a Ser/Thr proteinkinase complex that integrates signalsfrom nutrient availability, energy, andgrowth factors to regulate cell growth, pro-

liferation, and metabolism (1). Up-regulationof mTORC1 is associated with many diseasessuch as cancer, type 2 diabetes, and defects inneurodevelopment (1, 2). The mTORC1 com-plex is a dimer of mTOR/RAPTOR/mLST8heterotrimers (3). The mTORC1 kinase activ-ity is tightly regulated by two classes of smallGTPases, Rags and RHEB, both of which arenecessary for activation (4, 5). In responseto the abundance of nutrients, particularlyamino acids, active Rag heterodimers bindthe RAPTOR subunit of mTORC1 to recruitit to lysosomes (6–8), where mTORC1 can beallosterically stimulated by growth factor–activatedRHEB (3, 9–12). Unlike RHEB,whichcarries a C-terminal farnesylation that weaklyassociates it to a variety of membranes (13, 14),Rags have no lipid modification. Instead, theyassociate with the heteropentameric Ragulatorcomplex, which hasmyristoyl and palmitoylmod-ifications at the N terminus of its LAMTOR1subunit that localize it to lysosomes (15, 16).Recurrent oncogenic mutations in RagC en-hance its association with mTORC1, leadingto increased mTORC1 signaling (17–19).

Both Rags and RHEB are members of theRas-like superfamily of GTPases. However, un-like most members, Rags are obligate hetero-dimers, with RagA or RagB pairing with RagCor RagD (20). Analysis of the composite ge-nome of Lokiarchaeum revealed that RagGTPases have an archaeal origin closely re-lated to the Arf family of Ras-like GTPasesthat are involved in vesicular sorting (21), butthe mechanistic implication of this similaritywas not clear.Rag heterodimers have four possible

nucleotide-binding states but are active formTORC1 binding only when RagA or RagBis guanosine triphosphate (GTP)–bound andRagC or RagD is guanosine diphosphate(GDP)–bound (6, 7, 22). The GTPase domainscommunicate so that binding of GTP by onesubunit inhibits GTP binding and inducesGTP hydrolysis by the other subunit (22).The nucleotide states of Rags are regulatedby GTPase-activating proteins (GAPs) such asGATOR1 and folliculin (23–25) and guaninenucleotide exchange factors (GEFs) such asSLC38A9 and Ragulator (26, 27).To elucidate how human Rags interact with

mTORC1 and how RagC mutations activatemTORC1, we determined the structures anddynamics of RagA/RagC complexes in isola-tion and bound to mTORC1. The structuresrevealed nucleotide-dependent conformationalchanges in Rags that are required for mTORC1binding, enabling us to understand the mech-anism by which oncogenic Rag mutationsfacilitate association with mTORC1.

HDX-MS shows that RagA/RagC protects thea-solenoid of RAPTOR

To map RagA/RagC interactions with theRAPTOR subunit of mTORC1, we carried out

hydrogen/deuterium exchange mass spec-trometry (HDX-MS). For this we used RagA-Q66LGTP/RagC-T90NGDP containingmutationsthat increase mTORC1 association (6, 7, 17, 19).The RagA-Q66L switch II mutation (fig. S1)(28) impairs GTP hydrolysis and is a potentactivator of mTOR signaling, with mice bear-ing this mutation dying within 1 day of post-natal life (29). The RagC-T90Nmutation bindsonly GDP and is the most frequent and potentoncogenic mutation in RagC (17, 19).RagA/RagC heterodimers were monodis-

perse (fig. S2A) and formed a 1:1:1 complexwith RAPTOR (fig. S2, B and C). Most RAPTORpeptides that showed decreased HDX uponRagA/RagC binding mapped to a contiguoussurface in the region of residues 541 to 678,encompassing three adjacent helical repeatsof the RAPTOR a-solenoid (Fig. 1A, fig. S3,and table S1). There was also reduced HDXin the insertion before the last two helices ofthe RAPTOR a-solenoid (peptides 760 to 780and 805 to 812) and in the WD40 domainitself. On the Rag side of the interface, RagAswitch I was protected fromHDX by RAPTOR(Fig. 1B), whereas RagC switches were not(Fig. 1C and table S1). To understand thecontext of the HDX-MS–measured dynam-ics, we determined the structures of RagA/RagC heterodimers both free and bound tomTORC1.

High-resolution crystal structures of activeRagA/RagC heterodimers

HDX-MS identified RagC residues 1 to 34 ashighly flexible (fig. S2D). For crystallization,we truncated this region, producing a variantRagC(34-399) that bound RAPTOR the sameas full-length RagA/RagC (fig. S2E) and therebyenabled us to determine high-resolution crystalstructures of RagA/RagC.The crystal structure of RagA-Q66L/

RagC(34-399)-T90N at 2.6 Å resolution showeda compact arrangement of C-terminal CRD do-mains that mediate heterodimerization andN-terminal GTPase domains (Fig. 1D and tableS2). The overall fold of the GTPase domainsis similar to other Ras-like GTPases, with con-served loops (G1 to G5 motifs) that engagebound nucleotides, as well as regions thatchange conformation depending on whetherGTP or GDP is bound, known as switches (30).RagA is bound to GTP and has Mg2+ asso-ciated with the GTP g-phosphate, whereasRagC is bound to GDP and has no boundMg2+ (Fig. 1, E and F). Whereas RagA hasswitch I, interswitch, and switch II ordered(Fig. 1D and fig. S1), the RagC-T90NGDP GTPasedomain has no density for all of switch I andinterswitch strand b2 (residues 84 to 105) andall of switch II (116 to 130). The C-terminalCRDs have a roadblock fold consisting ofa central five-stranded antiparallel b sheetsandwiched between two a-helical layers (31).

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1MRC Laboratory of Molecular Biology, Cambridge CB2 0QH,UK. 2Whitehead Institute for Biomedical Research,Cambridge, MA 02142, USA. 3Department of Biology,Massachusetts Institute of Technology, Cambridge, MA02142, USA. 4Howard Hughes Medical Institute,Massachusetts Institute of Technology, Cambridge, MA02139, USA. 5Koch Institute for Integrative Cancer Research,Cambridge, MA 02139, USA. 6Broad Institute of MIT andHarvard, Cambridge, MA 02142, USA.*Present address: Astex Pharmaceuticals, Cambridge CB4 0QA, UK.†Corresponding author. Email: rlw@mrc-lmb.cam.ac.uk

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The whole complex has a pseudo–two-foldsymmetry, with the two GTPase domains closeto each other and their switches on oppositefaces of the complex (Fig. 1D).

The cryo-EM structure of mTORC1 bound toRagA/RagC

To understand how active RagA/RagC inter-acts with intact mTORC1, we used cryo–electron microscopy (cryo-EM). In order tostabilize mTORC1 bound to RagA-Q66LGTP/RagC-T90NGDP, we used chemical cross-linkingand expressed the RagA/RagC heterodimerwith RagC fused to another mTORC1-bindingprotein, PRAS40, which is largely disordered(fig. S4 and table S3). Using this mTORC1-RagA/RagC complex (fig. S5A), we generateda final reconstruction of mTORC1 at 4.1 Åresolution (figs. S5 and S6A and table S4).This reconstruction showed extra densityadjacent to the a-solenoid region of RAPTOR(fig. S6A) that HDX-MS identified as the RagA/RagC binding site. The TOSmotif from PRAS40(fused to RagC) contacts a groove between theRAPTORN-terminal conserved (RNC) and thea-solenoid of RAPTOR, as also observed byHDX-MS (fig. S4, C to E).Focused classification with signal subtrac-

tion (32) showed that about 9.5% of particleswere bound to RagA/RagC, correspondingto 90,809 particles, and reconstruction ofthe mTORC1-RagA-Q66LGTP/RagC-T90NGDP

complex at 5.5 Å resolution revealed densityfor the RagA/RagC into which we could read-ily fit our high-resolution RagA/RagC crystalstructure (Fig. 2 and fig. S6, B to D). RagA/RagC interacts with the convex surface of theRAPTOR a-solenoid (Fig. 3A). The GTPase-containing ends of the horseshoe-shapedRagA/RagC heterodimer are closest to theRAPTOR a-solenoid, with the CRDs pointingaway from RAPTOR (Fig. 2, B and C). TheRagA GTPase domain makes much more ex-tensive RAPTOR contacts than RagC, and theinterface agrees with our HDX-MS analysis(Fig. 3, A and B). The overall conformation ofmTORC1 bound to RagA/RagC heterodimersis nearly identical to the conformation of apo-mTORC1 (3).Three helices from RAPTOR (a24, a26, and

a29) in the region of residues 546 to 650of the RAPTOR a-solenoid make an extensivenetwork of interactions with switch I andinterswitch strand b2 of RagA-Q66LGTP (Fig.3A). The GTPase domain of RagCGDP formslimited interactions with RAPTOR, includinga contact of RagC-Asp185 at the N terminusof helix a5 with Thr680 in RAPTOR helix a31.Although the GTPase domains form most ofthe interface with RAPTOR, there are somecontacts with the CRD domains. These in-volve the C terminus of RagA helix a8 (Ser243

and Lys244) and the N terminus of RagC helixa8 (Gln280), which come close together and

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Fig. 1. The crystal structure of a RagA/RagC heterodimer and HDX-MS analysis of its interaction withRAPTOR. (A) HDX-MS–identified regions protected from HDX in the RAPTOR/RagA-Q66LGTP/RagC-T90NGDP

complex. Decreases in HDX (blue) of RAPTOR upon RagA/RagC binding are depicted on the RAPTORstructure (from PDB ID 6BCX). (B and C) Differences in HDX for RagA-Q66LGTP (B) and RagC-T90NGDP

(C) upon RAPTOR binding for all the peptides at 0.3 s in D2O. Decreases in HDX are depicted in shades ofblue, increases in shades of red. (D) The crystal structure of RagA-Q66LGTP/RagC(34-399)-T90NGDP,highlighting the ordered switches in RagAGTP compared with disordered switches in the RagC-T90NGDP

oncogenic mutant. (E) The conserved G motifs of RagA-Q66LGTP that make up the nucleotide binding pocket.(F) The 2mFo – DFc map (contoured at 1.2s) for the GTP of RagA and the GDP of RagC together withthe putative H bonds that they make to the G motifs. These are much more extensive for GTP than forGDP. Amino acid abbreviations: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu;M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; Y, Tyr.

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engage two adjacent structural elements ofRAPTOR, helix a29 (Thr639, Asn643, Met646)and the end of the long, mostly disorderedinsertion after a31.Mutations of RAPTOR residues in helices a26

(Trp593-Cys594 or Arg597-Asp598) anda29 (Thr634-Asp635-His636) that contact the RagA-Q66LGTPswitch I/interswitch greatly reduce bindingto RagA/RagC (Fig. 3C). Previously, RagA mu-tations were identified that prevent RAPTORinteraction (33), and they map to the interfacewith RAPTOR in our structure. We also deter-mined the cryo-EM structure of mTORC1-RagA-Q66LGTP/RagC-T90NGDP where RagA/RagCwas not covalently fused to PRAS40. Notethat in both cryo-EM structures, the RagA/RagC heterodimer interacts with RAPTORin the same manner (figs. S7 and S8).

Cancer-associated mutations in RagA/RagCaffect communication between GTPase domains

Cancer-associated mutations in RagC increasemTORC1 binding (17–19), and we wanted to

gain insights into the structural basis forthis effect. The mutations cluster in variousnucleotide-sensing elements of RagC: theP-loop (e.g., Ser75), switch I (e.g., Thr90), in-terswitch (e.g., Trp115, Asp116), and switch II(e.g., Pro118) (fig. S1). The RagC-T90N mutanthad switch regions disordered (Fig. 1D). Tosee whether this disorder is specific for theT90Nmutation, we also determined a crystalstructure of RagA-Q66LGTP/RagC-S75NGDP at2.5 Å resolution (table S2). The RagC-S75Nmutation in the P-loop impairs GTP bindingby eliminating the interaction of Ser75 withMg2+. The structures of RagC-S75NGDP andRagC-T90NGDP are very similar, except thatRagC-S75NGDP has helix a2 of switch I (resi-dues 86 to 93) ordered in one of the twoheterodimers in the crystal asymmetric unit(Fig. 4A); this finding suggests that S75Ndestabilizes but does not completely dis-order switch I. Hence, T90N causes a greaterperturbation in switch I than does S75N,consistent with themore potent phenotype of

the T90Nmutation in cells (17). The structureof the isolated wild-type RagC GTPase domainbound to a GTP analog [PDB ID 3LLU (34)]shows a completely ordered switch I and helixa2 that closely superimposes with this helix inRagC-S75NGDP. In the RagC-S75NGDP struc-ture, OG1 of the Thr90 side chain is close to theO2′ of the bound nucleotide (3.7 Å), so it islikely that the larger T90N substitution leadsto disorder of helix a2.RagA/RagC GTPase domain contacts can be

grouped into three sets (Fig. 4B). One set is atthe center of the interface where the G5motifsof the two domains meet, with RagA-Trp165

near the equivalent of this residue in RagC,Tyr221 (Fig. 1E and fig. S1). The second set in-volves interactions between RagA switch Ihelix a2 and the RagC loop immediately fol-lowing the G5 motif. In particular, there is awater-mediated interaction between RagA-Arg34 and the side chain of RagC-Asp222 (Fig.4B). The third set consists of interactionsbetween RagC switch I helix a2 and the RagA

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Fig. 2. Architecture of the mTORC1-RagA/RagC complex.(A) Schematic representation of mTORC1 components(mTOR, RAPTOR, and mLST8). (B) Overall cryo-EM–basedmodel of the mTORC1-RagA/RagC complex. The two orderedregions of the PRAS40 moiety of the fusion construct are alsoshown. (C) Three views of the mTORC1-RagA/RagC complex showRagA/RagC sitting on top of the RAPTOR a-solenoid, with theGTPase domains making most of the interactions.

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G4/a5 loop. This set is present in the complexwith RagC-S75NGDP where RagC-Glu

89makesa salt link with RagA-Arg137, and RagC-Phe92

contacts RagA loop residues 131 to 133 (Fig.4B), but is absent in the complex with RagC-T90NGDP, where RagC switch I is completelydisordered. This demonstrates that oncogenicmutations greatly perturb the interface be-tween the GTPase domains.

Rearrangements within the RagA/RagCheterodimer when bound to mTORC1

Comparing the crystal structure of free RagA-Q66LGTP/RagC-T90NGDP and the cryo-EMstructure of RagA/RagC bound to mTORC1reveals a shift in the interface between theGTPase domains by ~7 Å (Fig. 4C and movieS1). This creates a more open space betweenthe two GTPase domains, which in the freeRagA/RagC would be kept closer by interac-tions involving switch I helix a2. This mightexplain why the oncogenic RagC-T90N muta-tion, which has helix a2 disordered, bindsmore easily to RAPTOR, because the RagChelix a2/RagA interactions are already dis-ruptedbefore theheterodimerbinds toRAPTOR.A similar structural change could occur in theRagC-L91P mutant associated with follicularlymphomas (17). Residue Lys84 in the a1-a2loop of RagC switch I forms salt links withresidues Asp290 and Asp294 of helix a8 in theCRD (Fig. 4A). The lymphoma-associatedmuta-tion RagC-K84T (17) would likely disrupt thisinteraction, which could facilitate RagC re-arrangement relative to RagA as seen in thecomplex with mTORC1.

Comparison of the CRDs from the freeRagA/RagC with those in the mTORC1-bound RagA/RagC shows a small shift in the orientationof the two CRDs so that in themTORC1-boundform, the top surface of the CRDs that em-braces the GTPase domains is more splayed(fig. S9 andmovie S1). Comparison of the CRDdimer from the free RagA/RagC with the CRDdimer bound toRagulator (35) also shows shiftsbetween the two CRDs (fig. S9). Together theseresults indicate that the interface between theCRD domains has some flexibility. This couldbe exploited by interactors such as Ragulatorto exert changes on the GTPase domains viathe CRDs, which could contribute to the es-tablished role of Ragulator as a GEF forRagC (27).

Structural basis for relaying nucleotidebinding to the CRDs

Although only the RagAGTP/RagCGDP statebinds RAPTOR, the reverse, inactive state,RagAGDP/RagCGTP, is essential for terminat-ing mTORC1 activation. Furthermore, someRag interactors such as galectin-8 preferen-tially associate with this state (36). Therefore,a structural understanding of both states is im-portant. In the active heterodimer, nucleotide-sensitive elements in the GTPase domain ofeach Rag form contacts with its own CRD. InRagAGTP, switch I and the b2-b3 interswitchfirmly engage the CRD. The switch I interac-tion is at the center of the heterodimer in-terface, primarily with the CRD helix a8,whereas the b2-b3 interaction is at the outeredge of the CRD (Fig. 5A and fig. S10A). These

two interactions flank a central contact in-volving both nonswitch (a1 and a6) andswitch (b2-b3) contacts with the b7-b8 hairpinof the CRD. In the GTP-loaded state of RagA,the tip of the interswitch protrudes beyondthe GTPase domain and slips into a pocketon the surface of the CRD (Fig. 5A and fig. S10B).The interswitch tip bound in the CRD pocketmay be an element of the structural basis forthe “locked” state for RagA that was proposedon the basis of a kinetic study of the com-munication between GTPase domains in theRag heterodimers (22). In contrast, the inter-switch loop in RagCGDP is in a retracted posi-tion and partially disordered. In the structureof the isolated GTPase domain of GTP-boundRagC [PDB ID 3LLU (34)], the interswitchprotrudes beyond the GTPase domain in amanner equivalent to RagAGTP. This is accom-plished by a two-residue shift in the register ofstrand b3 relative to b1, such that residues 109to 115 in RagC-T90NGDP are in the positionsof residues 111 to 117 of the GTP-bound RagC(Fig. 5B). The extended interswitch in RagCGTPwould clash with its CRD, which suggests thata change in the relative orientations of theGTPase andCRDdomainswould be required toaccommodate GTP binding by RagC (fig. S10B).All of the contacts of the GTPase domainwiththe CRD constitute possible mechanisms fornucleotide binding to imprint onto the CRD.

Dynamics of the active and the reverse,inactive state of Rags

To gain a better understanding of the con-formational changes that occur in the reverse

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Fig. 3. Interface between RAPTORand the RagA/RagC complex.(A) Close-up views of RagA/RagC bindingto the RAPTOR subunit of mTORC1.The CRDs are shown as transparentsurfaces. RAPTOR helices contactingswitch I and interswitch of RagAare shown as cylinders. Spheres markRAPTOR-RagA/RagC interface residues.(B) View of the interface, illustratingregions with a decrease in HDX (blue)upon formation of the RAPTOR-RagA/RagC complex. (C) Mutational analysisof the binding interface. Strep-taggedwild-type RAPTOR (WT) and three differentRAPTOR mutants (with mutations toAla as indicated) were assayed for theirability to pull down RagA-Q66LGTP/RagC-T90NGDP in vitro. The pull-downefficiencies of RAPTOR mutants werenormalized to WT RAPTOR. Valuesare means from three independentexperiments; error bars denote SD.

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state, we tried but did not succeed in ob-taining diffracting crystals for this hetero-dimer. As an alternative strategy, we usedHDX-MS to examine differences in confor-mation between the active (RagA-Q66LGTP/RagC-T90NGDP) and reverse (RagA-T21NGDP/RagC-Q120LGTP) states (Fig. 5D and table S5).Overall, both Rags showed less HDX through-out the GTPase domains when GTP-boundcompared with GDP-bound, indicating a morecompact domain when bound to GTP. Fur-thermore, upon GTP binding to RagA, thereis a distinct protection in its CRD domain(e.g., in CRD helix a8, b7/b8 and the hinge

that are engaged with the GTPase domain),suggesting communication of the nucleotidestate to the CRD (Fig. 5D and movie S2).Residues making up a pocket on the surfaceof the RagA CRD that accommodate the inter-switch in GTP-bound RagA have increasedexchange in the reverse state, which we at-tribute to retraction of the RagA interswitch,exposing the RagA CRD pocket. Consistentwith this, the GDP-bound RagA interswitchhas increased HDX (displayed on the RagAGTP

crystal structure in Fig. 5D and on a RagAGDP

model in movie S2). For RagC, there is lesschange in HDX in the CRD upon GTP bind-

ing. Although the RagC interswitch showsGTP-dependent HDX protection, the pocketon the RagC CRD analogous to the RagApocket has only small changes in protection,so currently we do not know the position ofthe interswitch in the GTP-bound RagC.

Implications for yeast TORC1 signaling

Comparing the yeast Gtr1GTP/Gtr2GDP structure[PDB ID 4ARZ (37)] with human RagAGTP/RagCGDP indicates very large conformationaldifferences, both in switches and in the rel-ative orientations of GTPase domains, withthe Gtr2 GTPase domain rotated about 36°relative to the RagC GTPase domain (fig. S11A).The arrangement of the Gtr1/2 GTPase do-mains is not compatible with binding tomTORC1 in the same manner as RagA/RagC(fig. S11B). This suggests that the Gtr1/2 GTPasedomains may reorient in order to bind Kog1,the yeast homolog of RAPTOR. The very dif-ferent conformation of the Gtr2GDP switch Iregion and the extreme orientation of theGtr2 GTPase domain relative to RagC mayreflect a fundamental difference betweenRagC and Gtr2. This could explain why RagA-Q66L can complement a Gtr1-deficient strain,whereas neither wild-type RagC nor a GDP-bound mutant RagC could complement Gtr2deficiency in yeast (38). Despite these differ-ences, the binding site of Gtrs on Kog1 mapsto a similar region on the Kog1 a-solenoid (39).Given the role of the Rag heterodimers in

recruiting mTORC1 to lysosomes, the consti-tutive association of yeast TORC1 with thevacuole is surprising (5). A recent report ele-gantly showed that upon glucose starvation,yeast TORC1 forms inactive, vacuole-associatedhelical tubes named TOROIDs and that theTOROID formation is antagonized by activeRags (Gtr1GTP/Gtr2GDP) in cells (40). Fittingour RagA/RagC heterodimers into the cryo-EM reconstruction of the tubes, in accordancewith the arrangement present in our mTORC1-RagA/RagC complex, suggests that the Gtr1/2binding would not be compatible with theTORC1 arrangement in theTORIODs (fig. S11C).This might mean that Gtr1/2 binding coulddirectly regulate assembly or disassembly ofthe tubes to activate TORC1. Further work isneeded to test this structure-based proposal.

Discussion

RagA/RagC binding causes no conformationalchange in mTORC1, which suggests that therole of the Ragulator/Rags complex is to lo-calize mTORC1 to lysosomes where it can beallosterically activated by RHEB. Rag/RAPTORinteraction requires a GTP-loaded RagA, sothat RagA switch I and interswitch are or-dered, because they make most of the inter-actions with RAPTOR. A reverse state of Ragswith GDP-loaded RagA and GTP-loaded RagCdoes not bind RAPTOR as well (6, 7), because

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Fig. 4. Interactionsbetween GTPase domainsin the RagA/RagCheterodimer. (A) Compari-son of RagA-Q66LGTP/RagC-T90NGDP with RagA-Q66LGTP/RagC-S75NGDP,illustrating ordering of helixa2 in switch I of RagC-S75N. Superposition was onthe RagA subunit.(B) Three sets of interac-tions between RagAGTP andRagCGDP GTPase domains.(C) A change in the orien-tation of RagA/RagCGTPase domains in freeRagA/RagC relative toRagA/RagC bound tomTORC1. Superpositionwas on the RagA subunit.The view is similar to (B).

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RagAGDP would have the switch regions dis-ordered, whereas RagCGTP could not interactwith RAPTOR, because RagC residues analo-gous to RAPTOR-binding residues of RagA arenot conserved (fig. S12).The structures suggest how the nucleotide-

bound state of one GTPase domain is commu-

nicated both between subunits to the pairedGTPase domain and within a subunit to itsCRD (Figs. 4 and 5 and movie S2). First, con-sistent with communication between GTPasedomains (22), both RagA and RagC have helixa2 (in switch I) contacting the paired GTPasedomain and filling the space between them

(Fig. 4, A and B). Second, there are severalsets of interactions between the GTPase andCRD domains within a subunit that HDX sug-gests are dynamic and nucleotide-dependent,including switch I and the interswitch. Theinterswitch of Rag GTPases apparently under-goes a nucleotide-dependent register shift ofstrand b3 relative to b1 that could be part of amechanism to transmit nucleotide-bindinginformation from the GTPase domain to theCRD (Fig. 5). This is analogous to conforma-tional changes that accompany transition fromthe GDP- to GTP-bound states of Arf familyGTPases (Fig. 5C) (41, 42) and is consistentwith the evolutionary relationship of the Ragsto the Arf family (21). In Arfs, this interswitchtoggle between retracted and protruded con-formations coordinates membrane bindingwith GTP loading (Fig. 5B). In Rags, the inter-switch toggle could be part of a mechanismthat rotates one GTPase domain via a CRDfulcrum relative to the other GTPase domain.This would change RagA/RagC GTPase do-main contacts, making it less favorable forthe heterodimer to accommodate GTP in bothGTPase domains at the same time, as kineti-cally observed (22).mTORC1 activity is intricately regulated in

a signal- and location-specific manner. Mem-brane compartments act as signaling platformsthat serve to colocalize mTORC1 with its ac-tivating G protein RHEB, which is targetedtransiently to most endomembranes by far-nesylation (13). The lysosomal activity ofmTORC1 in amino acid signaling is achievedthrough its dynamic interface with the Rags-Ragulator lysosomal scaffold (8, 43). Ragscouple mTORC1 to lysosomes by binding toRAPTOR with their GTPase domains andto Ragulator with their CRDs. Because oflarge allosteric changes in mTOR that areincompatible with a mixed mTOR dimer,two RHEB molecules bind mTORC1 coop-eratively (3). In contrast, two soluble RagA/RagC heterodimers bind independently tomTORC1, because RagA/RagC binding doesnot introduce conformational changes inmTORC1.We propose an organization of active

mTORC1 on membranes based on our struc-ture of the mTORC1-RagA/RagC complex,the previously published structure of themTORC1-RHEB complex (3), and the crystalstructure of Ragulator bound to RagA/RagCCRDs (35, 44) (Fig. 6). In this model, RagA/RagC, associated with membranes throughRagulator (via the lipidated N terminus ofLAMTOR1), and RHEB, associated with mem-branes through C-terminal farnesylation, canbe bound at the same time to mTORC1, yetstill allow the mTOR active sites to face thecytosol. The RHEB-binding surface ofmTORC1would be near a RHEB-containing membrane,and the first ordered residue of the LAMTOR1

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RagA-Q66LGTP RagC-T90NGDPRagC-WTGMPPNP (3LLU)

RagCGDPCRD surface

W115

W115

A

2-residue shift

Arf6-GDP Arf6-GTPγS

GDP GTPγS

C

Mg2+

N-terminal helix

2-residue shift

GDP

GTP

β2 β3β3 β1

β1β2β3

RagCGDP RagAGTP

α6

8α8

α4α5

α4

α9

α7

α8

α7

α9

α6

α2

α8

α8

α2

α5α4

α5α4

α9α7

α7

α9

β1β3

α3

D

β3

β2

RagCGDPRagAGTP

HDX differences: [RagA-Q66LGTP/RagC-T90NGDP] - [RagA-T21NGDP/RagC-Q120LGTP]

GTPGDP

RagC-T90NGDP RagA-Q66LGTP

α2

α4α5

α6

α3α4

α6

α3’

GTPasedomain

CRDdomain

β1

α5

α4 α4

α2

B

( orange ribbon)

RagC-WTGMPPNP ( 3LLU, grey ribbon)

RagC-T90NGDP

Switch IInterswitch

InterswitchSwitch II

(RagA/C active - RagA/C inactive)

7

α2

α2

α5

β2

87

878 7

HDX exchange:

lessexchange

moreexchange

>12.06.0 to 12.02.0 to 6.0

-2.0 to -6.0-6.0 to -12>-12.0

+2.0 to -2.0

β1

β3α8

87

11

9 10

α2

α2

α4

α5

α1

α5

9 11

α1

10

α6

α3

β2

α1

180º

180º

Fig. 5. Structural basis for communicating nucleotide binding within the RagA/RagC heterodimer.(A) Both switch I and the interswitch make nucleotide state–dependent direct contacts with the CRD.(B) Superposition of GTP-bound RagC (PDB ID 3LLU, in gray) on the GDP-bound RagC (from our RagA-Q66LGTP/RagC-T90NGDP complex). The interswitch of GTP-bound RagC is black; GDP-bound RagC is red. TheTrp115 position illustrates a two-residue shift in strand b3 (relative to strand b1). The b2/b3 loop togglesbetween a retracted conformation in the GDP state and an extended conformation in the GTP state thatwould clash with the CRD if there were no conformational changes. (C) The structures of Arf6 bound to eitherGDP [PDB ID 1E0S (45)] or GTPgS [PDB ID 2J5X (46)], with switches colored as in (A). The interswitchtoggle couples nucleotide binding with membrane binding by the N-terminal helix. (D) Differences in HDXbetween the active (RagAGTP/RagCGDP) and inactive (RagAGDP/RagCGTP) states, illustrating changes in theCRDs in addition to the expected changes in the GTPase domains. In RagCGDP, disordered regions in theswitches have been modeled to illustrate all of the HDX changes.

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subunit of Ragulator (residue 96) would beabout 105 Å from the membrane surface,which suggests that the 95 flexible N-terminalLAMTOR1 residues could easily reach themembrane. Further structural and kineticanalysis ofmTORC1 complexes onmembraneswill be essential to fully appreciate the rolesof structural dynamics of mTORC1 with itsregulators and the roles of membranes inregulation of mTORC1.

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ACKNOWLEDGMENTS

We thank G. Cannone, A. Boland, S. Scheres, G. Murshudov,T. Nakane, R. Warshamanage, W. Zhang, P. Emsley, the MRC-LMBEM facility, and the LMB Scientific Computing team (J. Grimmettand T. Darling) for assistance and advice; S. McLaughlin, S. Maslen,F. Gorrec, and M. Yu from the LMB Facilities; A. Siebert for helpwith eBIC microscopes; S. Monaco and C. Mueller-Dieckmann forassistance with ESRF beamlines ID30B and ID30-A3; and DiamondLight Source for access to eBIC (funded by the Wellcome Trust,MRC, and BBSRC). Funding: Supported by a FEBS fellowshipand EMBO fellowship ALTF 1171-2016 (M.A.); a St. Catharine’s

Anandapadamanaban et al., Science 366, 203–210 (2019) 11 October 2019 7 of 8

Fig. 6. Model of the mTORC1-RHEB-RagA/RagC-Ragulator complex. (A) A ribbondiagram of the mTORC1-RHEB-RagA/RagC-Ragulator complex. RAPTOR-RagA/RagC was superimposed on RAPTOR in mTORC1-RHEB complex [PDB ID 6BCU (3)].The CRD of the mTORC1-RagA/RagC-RHEB complex was superimposed onto the

CRD of the crystal structure of the Ragulator-CRD domain complex [PDB ID 6EHR(35)]. (B) Expanded view of the Ragulator/Rags/RAPTOR interface. (C) A modelof the complex on a lysosomal membrane. The lipid-modified regions of LAMTOR1and RHEB that anchor them to membranes are depicted in arbitrary conformations.

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College fellowship (G.R.M.); a junior fellowship from TuberousSclerosis Association, a researcher mobility grant from the RoyalSociety of Chemistry, and a travelling fellowship from the Companyof Biologists (K.B.R.); NIH grants R01 CA103866, R01 CA129105,and R37 AI047389, Department of Defense grant W81XWH15-1-0230, and the Lustgarten Foundation (D.M.S.); and MedicalResearch Council grant MC_U105184308 and Cancer ResearchUK grant C14801/A21211 (R.L.W.). D.M.S. is an HHMI investigatorand an American Cancer Society Research Professor. Authorcontributions: M.A., G.R.M., O.P., A.B., J.K., C.M.J., and K.B.R.conducted the research. M.A., G.R.M., A.B., B.S., and R.L.W.

analyzed data. M.A., O.P., D.M.S., and R.L.W. developed theexperimental plan. M.A. and R.L.W. wrote the draft. All authorsreviewed and edited the drafts. Competing interests: Authorsdeclare no competing interests. Data and materials availability:Materials supporting findings are available from R.L.W. uponreasonable request. Coordinates for crystal structures ofRagA/RagC GTPase heterodimers were deposited in theProtein Data Bank, accession codes 6S6A and 6S6D. Thecryo-EM reconstructions of the mTORC1-RagA/RagC complexwas deposited with EMDB (EMD-10132 and EMD-10133) andPDB (entries 6SB0 and 6SB2).

SUPPLEMENTARY MATERIALS

science.sciencemag.org/content/366/6462/203/suppl/DC1Materials and MethodsFigs. S1 to S13Tables S1 to S5Movies S1 and S2References (47–75)

View/request a protocol for this paper from Bio-protocol.

21 March 2019; accepted 3 September 201910.1126/science.aax3939

Anandapadamanaban et al., Science 366, 203–210 (2019) 11 October 2019 8 of 8

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Architecture of human Rag GTPase heterodimers and their complex with mTORC1

Balaji Santhanam, Kacper B. Rogala, David M. Sabatini and Roger L. WilliamsMadhanagopal Anandapadamanaban, Glenn R. Masson, Olga Perisic, Alex Berndt, Jonathan Kaufman, Chris M. Johnson,

DOI: 10.1126/science.aax3939 (6462), 203-210.366Science 

, this issue p. 203Scienceconsistent with the idea that mTORC1 must sense additional growth regulators before it is activated.RagC subunits in the heterodimer. RagA/RagC binding causes no conformational change in mTORC1, which is required for binding to mTORC1 and support a mechanism for conformational communication between the RagA andstructures of a RagA/RagC heterodimer. The structures, together with dynamic studies, explain the nucleotide states

electron microscopy and crystal− determined cryoet al.converge on the mTORC1 complex. Anandapadamanaban small guanosine triphosphatase, bind to mTORC1 and recruit it to the lysosome. Here, other signaling pathwaysrole in integrating multiple signals to regulate cell growth. When nutrients are abundant, heterodimers of Rag, a class of

The mechanistic target of rapamycin complex 1 (mTORC1) is known as the master kinase, acknowledging its keyMastering regulation

ARTICLE TOOLS http://science.sciencemag.org/content/366/6462/203

MATERIALSSUPPLEMENTARY http://science.sciencemag.org/content/suppl/2019/10/09/366.6462.203.DC1

REFERENCES

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