Abstract
The mammalian target of rapamycin (mTOR) is a large, multidomain protein kinase, which plays a central role in the regulation of cell growth and has recently emerged as an essential target of survival signals in many types of human cancer cells. Here, we report the solution structures of complexes formed between the FKBP12-rapamycin binding (FRB) domain of mTOR and phosphatidic acid, an important cellular activator of the kinase, and between the FRB domain and a novel inhibitor (HTS-1). The overall structure of the FRB domain is very similar to that seen in the ternary complex formed with FKBP12 and the immunosuppressive drug rapamycin; however, there are significant changes within the rapamycin-binding site with important consequences for rational drug design. The surface of the FRB domain contains a number of distinctive features that have previously escaped attention, including a potential new regulatory site on the opposite face to that involved in the binding of rapamycin, which displays the features expected for a specific binding site for a small molecule. The interaction sites for phosphatidic acid and HTS-1 were found to closely match the site responsible for rapamycin binding. In addition, the structures determined for the FRBphosphatidic acid and FRB-HTS-1 complexes revealed a striking similarity between the conformations of buried portions of the ligands and that seen for the rapamycin backbone in contact with the domain. Our findings further highlight the importance of the FRB domain in small molecule-mediated regulation of mTOR, demonstrate the ability to identify novel inhibitors of mTOR that bind tightly to the rapamycin-binding site in the absence of FKBP12, and identify a potential new regulatory site that may be exploited in the design of new anticancer drugs.
Introduction
The mammalian target of rapamycin (mTOR) is a relatively large protein kinase (2549 residues), which plays a central role in the regulation of cell growth and has recently been shown to be a critical target of survival signals in cancer cells. The protein is highly conserved across all eukaryotes, with 40-60% amino-acid sequence identity and is a member of the phosphatidylinositol kinase-related kinase (PIKK) family (for recent reviews see Guertin and Sabatini, 2005; Sarbassov et al., 2005; Wullschleger et al., 2006; Foster, 2007) . mTOR contains a number of distinct functional domains including up to 12 HEAT repeats in the N-terminal region, central FAT (Bresidues 1513 -1910 , FRB (B2015-2114) and serinethreonine protein kinase domains (B2181-2484) and a C-terminal FATC domain (B2517-2549). The FRB domain of mTOR is responsible for the binding of the inhibitory cyclic macrolide rapamycin in complex with the small peptidyl-prolyl cis-trans isomerase FKBP12 (107 residues (Vezina et al., 1975; Heitman et al., 1991; Kunz et al., 1993; Brown et al., 1994; Sabatini et al., 1994; Choi et al., 1996) ). The precise mechanism by which binding of the complex can inhibit the function of mTOR is poorly understood, but is probably related to its ability to cause a reduction in the catalytic activity of the kinase domain, as demonstrated by the affect of rapamycin on the phosphorylation of specific substrates of mTOR such as p70S6 kinase (1-3). Rapamycin has proved to be an important immunosuppressive drug as well as a very useful tool to study the cellular roles of mTOR, and future drugs targeted at the rapamycinbinding site on mTOR appear to have great potential in cancer treatment (Guertin and Sabatini, 2005; Petroulakis et al., 2006; Thomas, 2006) .
Introduction
Recently, mTOR has been shown to be present in two distinct protein complexes in cells, termed mTORC1 and mTORC2 (Hara et al., 2002; Kim et al., 2002; Loewith et al., 2002; Jacinto et al., 2004; Sarbassov et al., 2004 Sarbassov et al., , 2005 Guertin and Sabatini, 2005; Wullschleger et al., 2006) , which both contain mTOR and a known binding partner mLST8/GbL (260 residues), but differ in that the third protein component of mTORC1 is raptor (1335 residues) and of mTORC2 is rictor (1708 residues). The well characterized rapamycin-sensitive functions of mTOR, such as the activation of translation and inhibition of autophagy, have been shown to be associated with mTORC1 (Hara et al., 2002; Kim et al., 2002) , which is consistent with the inability of mTORC2 to bind rapamycin (Loewith et al., 2002; Sarbassov et al., 2004) . mTORC2 is clearly involved in regulating the assembly of the actin cytoskeleton in cells (Jacinto et al., 2004; Sarbassov et al., 2004 ) and also appears to be a key activator of the protein kinase Akt (Sarbassov et al., 2005) . This is an essential component of the insulin/phosphatidylinositol 3-kinase (PI3K) signalling pathway and an attractive drug target in many human cancers, such as those associated with loss of function of the tumour suppressor protein phosphatase and tensin homologue deleted on chromosome band (PTEN), resulting in hyperactive signalling via Akt (Guertin and Sabatini, 2005; Bianco et al., 2006) . There is also evidence to suggest that Akt indirectly activates mTORC1 via phosphorylation-induced inhibition of the complex formed by the tuberous sclerosis (TSC) proteins TSC1 and TSC2, which acts as a negative regulator of mTORC1 activity (Hay and Sonenberg, 2004; Manning, 2004) . This provides a crucial link between the regulation of cell growth, division and survival and interestingly places mTORC1 as a downstream effector of mTORC2.
Introduction
The activity of mTOR is known to be modulated by a number of small ligands including rapamycin, phosphatidic acid and amino acids such as leucine. Rapamycin and phosphatidic acid have both been shown to bind to the FRB domain of mTOR (Choi et al., 1996; Fang et al., 2001; Avila-Flores et al., 2005) , perhaps suggesting a general role for this domain in ligand-mediated regulation of the kinase. Recent findings suggest that the potent inhibitory affect of rapamycin may arise from blocking of the activation of mTOR by phosphatidic acid binding, either by direct competition for overlapping interaction sites, or by an allosteric mechanism (Chen and Fang, 2002; Foster, 2007) . For example, increased activity of the phospholipase responsible for phosphatidic acid production in human breast cancer cells has been shown to reduce their sensitivity to rapamycin (Chen et al., 2003) . There is still considerable uncertainty concerning the mechanism of the regulation of mTOR by amino acids; however, several recent studies suggest that amino acids may act directly on mTORC1, implying a specific binding site within the complex (Kim et al., 2002; Long et al., 2005; Sarbassov et al., 2005; Wullschleger et al., 2006) .
Introduction
In this communication, we report the structure of the regulatory complex formed between the FRB domain of mTOR and phosphatidic acid, as well as the structure of the complex formed between the FRB domain and a novel class of mTOR inhibitor identified from a high-throughput screen (HTS). Analysis of the surface features of the FRB domain also reveal the presence of a potential new regulatory site on the opposite face to that involved in binding to rapamycin and phosphatidic acid, which could accommodate an amino acid such as leucine. This work further highlights the importance of the FRB domain in ligand-mediated regulation of mTOR, clearly demonstrates the ability to identify novel inhibitors of mTOR that bind tightly to the rapamycin-binding site in the absence of FKBP12, and identifies further potential regulatory sites that may be exploited in the design of new drugs.
Results and discussion
Solution structure of the mTOR FRB domain Despite a limiting solubility of about 150 mM nearly complete 15 N-, 13 C-and 1 H-resonance assignments were obtained for the FRB domain (Veverka et al., 2006) , which allowed automated assignment of the nuclear overhauser effect (NOE) peaks identified in threedimensional 15 N-and 13 C-edited NOESY and in the aromatic to aliphatic region of two-dimensional NOESY spectra using the CANDID procedure implemented in CYANA (Herrmann et al., 2002 H HMQC-NOESY and NOESY spectra, respectively. In the final round of structural calculations, 32 satisfactorily converged FRBdomain structures were obtained from 100 random starting conformations using a total of 1861 nuclearmagnetic resonance (NMR)-derived structural constraints (an average of 18.6 constraints per residue for the welldefined region corresponding to residues 2019-2114). The distribution of NMR constraints and structural statistics for the FRB domain are summarized in Table 1 . The FRB-domain structures, together with the NMR constraints, have been deposited in the Protein Data Bank under accession number 2NPU. Structures of regulatory complexes formed by the FRB domain V Veverka et al
Results and discussion
The solution structure of the mTOR FRB domain has been determined to relatively high precision, which is clearly apparent from the overlay of the protein backbone shown for the family of converged structures in Figure 1a . This is also reflected in fairly low-root meansquared deviation (r.m.s.d.) values to the mean structure for both the backbone and all heavy atoms (0.8270.25 and 1.6170.27 Å , respectively) of residues forming the well-defined region (residues 2019-2114). The FRB domain contains a disordered, flexible N-terminal region (residues 2014-2018), but the remaining residues form a well-defined structure consisting of four helices (a1: W2023-G2040, a2: V2044-R2060, a3: L2065-L2090 and a4: V2094-S2112) joined by short loops that collectively form a four-helix bundle (Figure 1b) . The third helix contains a marked bend of about 451 involving residues W2074-R2076. During the preparation of this manuscript a fairly low-resolution solution structure was reported for the isolated-FRB domain (Leone et al., 2006) , which was based on less than half the number of constraints used to determine the structure reported here and probably reflects the poor stability and solubility observed for the shorter polypeptide used in that study (100 residues). For the family of 19 FRB structures reported (Leone et al., 2006) , the r.m.s.d.s to the mean for backbone and all heavy atoms are 1.3070.33 and 2.1870.35 Å , respectively for residues 2019-2114, which is significantly greater than for the FRB-domain structures described here (see above). If only residues in the helical regions are considered, the backbone atom r.m.s.d. falls to 1.0370.32 Å , as compared to 0.5270.13 Å for the FRB structures reported here. The overall topology of the lower resolution structure is very similar to that described here apart from the N-terminal half of helix 3 (L2065-D2077), which is largely disordered. In addition, many of the side chains forming the rapamycin-binding site were reported to be poorly defined by the limited NMR data (Leone et al., 2006) .
Results and discussion
Analysis of the surface of the high-resolution structure obtained for the FRB domain revealed a number of notable features, including a shallow hydrophobic patch formed by the side chains of residues from helices a1 and a4 (L2031, F2039, W2101, Y2105 and R2109; Figure 1c ), which has been shown to bind rapamycin in the complex formed with FKBP12 (Choi et al., 1996) . There is also an extensive hydrophobic strip between helices a1 and a2 that appears well suited to binding the nonpolar face of a helix and in the intact protein may form the interaction surface with either another domain of mTOR, or one of its functional partner proteins. The opposite face of the FRB domain to that involved in rapamycin binding contains a striking deep cleft between helices a2 and a3, which spans almost the entire length of the domain and is lined by a mixture of charged and hydrophobic groups ( Figure 1d ). This cleft displays the features expected for a specific ligandbinding site on a protein and it seems probable that it forms the binding site for a small molecule regulator of mTOR. In terms of size and shape the putative regulatory site could accommodate leucine, which is a known regulator of mTOR activity (Kim et al., 2002; Long et al., 2005; Sarbassov et al., 2005; Wullschleger et al., 2006) . However, in NMR titration experiments we were unable to obtain any evidence for leucine binding to the isolated-FRB domain.
Comparison of the free and rapamycin/FKBP12-bound FRB domain
The solution structure of the isolated-FRB domain is very similar to that observed in the complex with rapamycin and FKBP12, which is reflected in the backbone and all heavy atom r.m.s.d. values of 1.1570.50 and 1.7770.77 Å , respectively, for a best-fit superposition of the well-defined region (residues 2019-2114). However, there are significant differences in the shape of the rapamycin-binding site arising from changes in the orientations of amino-acid side chains. In the isolated-FRB domain, the binding site consists of two fairly shallow pockets linked via a narrow cleft Structures of regulatory complexes formed by the FRB domain V Veverka et al formed by the aromatic rings of F2039 and Y2105 ( Figure 1c ). In contrast, with rapamycin bound, the site is essentially elliptical in shape and somewhat shallower, with the narrow cleft lost due to rearrangements of the side chains of F2039 and Y2105 required to accommodate the binding of rapamycin. The localized changes in side-chain conformations induced by rapamycin binding clearly illustrate the inherent limitations of rigid-body-based methods for either correctly docking ligands into known binding sites on proteins, or for predicting the types of small molecules able to bind to specific sites on proteins, and highlights the potential importance of the general approach described below. Figure 3a . The binding of phosphatidic acid clearly results in very significant shifts in the positions of backbone amide signals from two distinct groups of residues located towards the N and C terminus of the FRB domain, in particular, E2032, E2033, R2036, L2037, F2039, Q2099, A2100, Y2105, H2106 and F2108. These residues form a distinct patch on the surface of the domain (Figure 4a ), which closely matches the region involved in rapamycin binding (Choi et al., 1996; Banaszynski et al., 2005) and clearly indicates substantial overlap of the rapamycin and phosphatidic acid-binding sites. Previous studies have proposed that the side chain of R2109 plays a key role in phosphatidic acid binding by interacting with the negatively charged phosphate group (Fang et al., 2001) . Signals from the backbone amide group of R2109 were not significantly perturbed by phosphatidic acid binding (Figure 3a) . However, we did observe a change in the position of the signals ( 15 N and 1 H) from the side-chain amino group of R2109 (Dd ¼ 0.034 p.p.m.), which is clearly consistent with its proposed role in phosphatidic acid binding. Previous evidence for a specific interaction between the FRB domain and phosphatidic acid was based on the finding that FRB bound to small unilamellar vesicles containing as little as 10% 1-palmitoyl 2-oleoyl phosphatidic acid (Fang et al., 2001 ). This interaction was also shown to be inhibited by the addition of equimolar amounts of Structures of regulatory complexes formed by the FRB domain V Veverka et al the GST-FKBP12-rapamycin complex. A significant advantage of the NMR approach described here is that it provides direct and unambiguous evidence for the binding of phosphatidic acid to a specific site on the FRB domain, which can be used to determine the structure of the complex formed. In addition, from comparison of the 15 N/ 1 H HSQC spectra of the free and phosphatidic acid-bound FRB it is clear that no significant conformational change is induced in the domain by binding of the ligand.
Comparison of the free and rapamycin/FKBP12-bound FRB domain
The significant shifts in specific backbone and sidechain NH signals induced by phosphatidic acid binding were used to generate structures of the complex consistent with the NMR data using the ambiguous interaction restraints (AIRs)-based docking protocol implemented in the program HADDOCK (Dominguez et al., 2003) . Visual inspection of the solution structure of the FRB domain identified a patch of five residues with substantially solvent exposed side chains and significantly perturbed NMR signals (E2032, F2039, Y2105, H2106 and R2109), which were included as active residues in the docking procedure. A further three surface residues located adjacent to this group and with the potential to contribute to phosphatidic acid binding (L2031, S2035 and W2101) were defined as passive residues. In addition, a specific-distance constraint was included to impose the interaction between the side chain of R2109 and the phosphate group of the ligand, which is supported by the NMR data and previous mutagenesis studies (Fang et al., 2001) . The docking calculations produced only a single family of structures for the FRB-domain-phosphatidic acid complex, as illustrated in Figure 5a , which are entirely consistent with the NMR data and have favourable energy terms. 
Structures of regulatory complexes formed by the FRB domain V Veverka et al
This is somewhat surprising given the inclusion of only a single unambiguous docking constraint, but probably reflects the restrictions imposed by the clear amphipathic nature of the ligand (highly polar head group and hydrophobic tail) and the features of a well-defined binding site. As expected, the phosphate group of phosphatidic acid binds to a positively charged patch formed by the side chain of R2109. One of the acyl chains occupies part of the rapamycin-binding site and makes favourable van der Waals interactions with the side chains of L2031, F2039, W2101 and Y2105, while the other binds to a neighbouring region and contacts the side chains of D2102, Y2105 and H2106 (Figure 5b ). The partial overlap between the phosphatidic acid and rapamycin-binding sites on the FRB domain clearly suggests that rapamycin will inhibit the activation of mTOR by phosphatidic acid, and provides further support for the proposal that this may be the principle mechanism of rapamycin-induced inhibition of mTOR (Chen and Fang, 2002; Foster, 2007) . The interaction between phosphatidic acid and mTOR presumably occurs at a membrane surface, as recently reported for upstream activators of Ras (Hancock, 2007; Mor et al., 2007; Zhao et al., 2007) , which will limit the FRB domain's accessibility to the hydrophobic acyl chains of phosphatidic acid. This probably accounts for the relatively tight and specific interaction observed with the short acyl chains form of phosphatidic acid, which essentially occupies the entire hydrophobic-binding site on the FRB domain ( Figure 5 ). H HSQC spectra shown in Figure 2 and for fully HTS-1-bound FRB are summarized by the histogram in Figure 3b . The interaction with HTS-1 clearly results in significant shifts in the positions of backbone amide signals from two distinct groups of residues, which are again located towards the N-and C terminus of the FRB domain, including E2033-R2036, Y2038-N2043, T2098, A2100, W2101 and Y2104-R2110. As illustrated in Figure 4b , these residues form a contiguous patch on the surface of the domain, which closely matches the region involved in both phosphatidic acid and rapamycin binding (Choi et al., 1996; Banaszynski et al., 2005) . Comparisons of the shifts induced in backbone amide signals by the addition of phosphatidic acid, HTS-1 or the two combined, clearly indicate that the two ligands compete for binding to the same site on the FRB domain, as illustrated in Figure 6 . The HTS-1-binding site contains a network of six aromatic residues (Y2038, F2039, W2101, Y2104, Y2105 and F2108), which apart from Y2104 make direct van der Waals contacts with rapamycin in the ternary FKBP12-rapamycin-FRB complex (Choi et al., 1996) .
Structures of regulatory complexes formed by the FRB domain V Veverka et al
The chemical shift perturbation data obtained for the binding of HTS-1 was used as AIRs in docking calculations designed to assess the diversity of FRB-HTS-1 complex structures consistent with the NMR data, with the surface residues E2032, S2035, Y2038, F2039, T2098, W2101, Y2105 and F2108 defined as active and H2028 and L2031 as passive (Dominguez et al., 2003) . In contrast to phosphatidic acid, four distinct modes of HTS-1 binding were found to be compatible with the induced chemical shift changes, which all featured an extensive network of hydrophobic interactions between the ligand and residues within the rapamycin-binding site of the FRB domain. The four possible structures for the complex were comparable in terms of both intermolecular energy values (À46.373.1 H-HSQC spectra of uniformly 15 N-labelled FRB (100 mM) and illustrate the shifts induced by the addition of a 10-fold molar excess of phosphatidic acid (red), HTS-1 (green) or the two combined (blue). In all panels, the peak observed in spectra of the free FRB domain is shown in black for reference. þ and OH). Acquisition of a series of NOESY spectra from the complex with mixing times ranging from 100 to 1600 ms revealed that peaks arising from NOEs between backbone amides separated by up to 8 Å reached a maximum with a mixing time of 1300 ms, which is consistent with previous reports (Koharudin et al., 2003) . These spectra also contained a few peaks corresponding to very long-range NOEs between backbone amides of FRB and groups on HTS-1, which are listed in Table 2 .
Structures of regulatory complexes formed by the FRB domain V Veverka et al
The inclusion of intermolecular distance constraints reflecting both the chemical shift perturbation and intermolecular NOE data in docking calculations produced only a single family of converged structures for the FRB-HTS-1 complex, as illustrated in Figure 7a . HTS-1 bound to the FRB domain adopts a very similar position to that seen for rapamycin in the FKBP12-rapamycin-FRB complex (Figure 7b) , with the buried portion of the ligand mimicking the conformation of the rapamycin backbone involved in direct contacts with FRB. Van der Waals interactions are seen between HTS-1 and the side chains of H2028, L2031, E2032, S2035, Y2038, F2039, T2098, W2101, Y2105 and R2109, which includes all but one of the residues (F2108) involved in hydrophobic contacts with rapamycin (Choi et al., 1996) . The FRB-HTS-1 complex is clearly stabilized by several favourable interactions between aromatic rings on the protein and ligand. For example, the central pyrimidine ring of HTS-1 makes p-p interactions with the phenyl ring of Y2105 and the thiophene group on the ligand makes similar interactions with the indolic ring of W2101, as illustrated in the expanded view of the HTS-1-binding site shown in Figure 7c . The interactions seen in the complex also provide explanations for preliminary structure activity relationships derived for the HTS-1 ligand family, such as the absolute requirement for a terminal benzyloxy group linked to a cyclopentyl or cyclohexyl ring. HTS-1 and rapamycin have completely unrelated chemical structures, but show many similarities in their interactions with the FRB domain. This suggests that it may be possible to develop novel small molecule therapeutics that mimic the action and potency of rapamycin but have more desirable properties.
Conclusions
The detailed structural information obtained for the FRB-HTS-1 complex, together with comparable information for the FRB-phosphatidic acid complex, clearly highlights the potential of combining readily accessible NMR chemical shift perturbation data with AIRs-based docking to determine reliable structures for proteinligand complexes. In many cases, the inclusion of a (9) is illustrated, with the domain shown in the same orientation as in panel a. In (c), the relative orientations of HTS-1 and key interacting residues are highlighted, with covalent bonds colour coded as described in Figure 4 .
Conclusions
Structures of regulatory complexes formed by the FRB domain V Veverka et al relatively small number of more specific-distance constraints, such as those derived from very long-range intermolecular NOEs identified in spectra of perdeuterated protein samples, will be sufficient to determine a unique solution for the complex, as seen for FRB-HTS-1. The approach described in this communication should be applicable to many potential pharmaceutical targets and with further refinement should be sufficiently rapid and reliable to guide medicinal chemistry programmes designed to optimize the properties of candidate drug molecules. The determination of both the site and mode of interaction of phosphatidic acid with the FRB domain of mTOR clearly confirms its potential importance as an activator of the kinase and supports recent suggestions that rapamycin may exert its inhibitory affect on mTOR by blocking phosphatidic acid-mediated activation (Chen and Fang, 2002; Foster, 2007) . There is currently no evidence to suggest that either activators or inhibitors of mTOR that bind to the FRB domain induce a significant conformational change, which could modulate the activity of the kinase. In addition, the majority of the interactions with both classes of ligands involve the same residues on the FRB domain (L2031, E2032, F2039, W2101, Y2105 and R2109). This suggests that the phosphatidic acid-mediated localization of mTOR to a membrane surface may be the basis for activation, as recently reported for upstream activators of Ras (Hancock, 2007; Mor et al., 2007; Zhao et al., 2007) . The work reported here also reveals that the surface of the FRB domain contains a number of distinctive features that have escaped previous attention, including an extensive hydrophobic strip between helices a1 and a2 that appears well suited to forming a proteinprotein interface and a potential binding site for a regulatory small molecule on the opposite face to that involved in interactions with rapamycin and phosphatidic acid. This clearly suggests an essential role for the FRB domain in mediating specific interactions with both regulatory small molecules and protein domains from either mTOR or functional partners. The structural studies reported here clearly highlight the importance of the FRB domain in small molecule-mediated regulation of mTOR and may hint at potential new avenues for therapeutic intervention, perhaps involving exploitation of the putative new regulatory site.
Materials and methods
Expression and purification of the mTOR FRB domain Samples of a 126 residue protein corresponding to the FRB domain from mTOR (residues 2014-2114) with an N-terminal histidine tag were prepared as described previously using an Escherichia coli-based expression vector (Veverka et al., 2006 NMR spectroscopy NMR spectra were acquired from 0.35 ml samples of the FRB domain (100-150 mM) in a 25 mM sodium phosphate and 100 mM sodium chloride buffer at pH 7.0. All the NMR experiments were collected at 251C on a 600 MHz Bruker Avance spectrometer (Bruker BioSpin GmbH, Germany) equipped with a triple-resonance ( 15 N/ 13 C/ 1 H) cryoprobe. A series of double and triple-resonance spectra were recorded to determine essentially complete sequence-specific resonance assignments for the FRB domain, as described previously (Veverka et al., 2006 (Koharudin et al., 2003) . The 1 H signals from free ligands were assigned to specific groups using correlations observed in TOCSY and NOESY spectra, and as the ligands investigated were in fast exchange on the NMR timescale between the free-and FRB-bound states these assignments could be readily transferred to the bound ligands by following the movement of the relevant signals in NMR titration experiments. The two-dimensional and three-dimensional spectra were acquired, processed and analysed as described previously (Veverka et al., 2006) .
Protein structural calculations
The family of converged FRB-domain structures was calculated in a two-stage process using the programme Cyana 1.0.6 (Herrmann et al., 2002) . Initially, the combined automated NOE assignment and structure determination protocol (CAN-DID) was used to automatically assign the NOE cross-peaks identified in two-dimensional NOESY and three-dimensional 15 N-and 13 C-edited NOESY spectra, and to produce preliminary structures. Subsequently, several cycles of simulated annealing combined with redundant dihedral angle constraints (REDAC) were used to produce the final converged FRBdomain structures (Guntert and Wuthrich, 1991) . Analysis of the family of structures obtained was carried out using the programs Cyana, Molmol, PyMol and Procheck (Koradi et al., 1996; Laskowski et al., 1996; DeLano, 2002; Herrmann et al., 2002) .
Analysis of NMR ligand-binding data
The minimal shift approach (Farmer et al., 1996; Williamson et al., 1997; Muskett et al., 1998) N-chemical shifts between pairs of compared HSQC peaks and a N is a scaling factor of 0.2 required to account for differences in the range of amide proton-and nitrogen chemical shifts. For each individual backbone amide peak, the minimal shift induced by ligand binding was taken as the lowest calculated combined shift value (Dd).
Analysis of NMR ligand-binding data
To facilitate the identification of ligand-binding sites on the surface of the mTOR FRB domain, histograms of minimal combined shift (Dd) versus the protein sequence were used to reveal regions of the protein containing a number of significantly perturbed backbone amide signals. The affected residues within these regions were then assessed as possible contact points in the ligand-binding site by examination of the solution structure determined for the FRB domain. In this analysis, only clusters of residues located on the surface of the protein were considered to be available for ligand binding.
Calculation of FRB-ligand complex structures by NMR constraint-driven docking
The software package HADDOCK (high-ambiguity-driven docking, Dominguez et al., 2003) was used to determine possible orientations and conformations for small ligands binding to NMR-identified sites on the surface of the FRB domain. In this recently developed approach, the algorithm guiding the docking of the protein and ligand is driven by a combination of ambiguous distance constraints derived from combined minimal shift data (Dd) and if available specific 1 H-1 H distance constraints obtained from intermolecular NOEs (protein ligand). The docking protocol consists of three distinct stages: randomization of the relative orientations of the two binding partners and energy minimization of each, semi-rigid-body docking of the protein and ligand using a simulated annealing protocol, and finally refinement of the complexes obtained by molecular dynamics in a water shell.
Calculation of FRB-ligand complex structures by NMR constraint-driven docking
In the first stage, the protein and ligand were typically placed about 15 Å apart, randomly rotated with respect to each other, energy minimized and from 1000 starting combinations, the 200 lowest energy solutions were selected for further refinement. The next stage involved three rounds of simulated annealing-based docking, in which the protein was initially maintained as a rigid body with subsequent cycles allowing the side chains and then all atoms of residues at the ligand-binding site to move. The small molecule ligands were defined as fully flexible during these steps. The final stage of the docking procedure consisted of refinement of the proteinligand complexes by molecular dynamics in an 8 Å water shell. The complex structures obtained were grouped into clusters on the basis of r.m.s.d. values for the backbone atoms of protein residues at the binding site and for the bound ligand. Typical clusters contained an ensemble of at least 10 complexes with a backbone r.m.s.d. of less than 1.0 Å and were ranked according to their average interaction energy (Dominguez et al., 2003) .
Calculation of FRB-ligand complex structures by NMR constraint-driven docking
The family of converged solution structures obtained for the FRB domain, together with ligand molecules parameterized with the XPLO2D Software (Kleywegt and Jones, 1997) , were used as an input for the docking calculations. Ambiguous distance constraints were defined between residues on the FRB domain involved in ligand binding (identified by the minimal shift approach) and all ligand atoms. Intermolecular NOEs were included in the docking calculations as upper distance limits of up to 8.0 Å between specific backbone amide protons of the FRB-domain and -ligand protons.

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