Activating mutations in TOR are in similar structures as oncogenic mutations in PI3KCalpha

Abstract

TOR (Target of Rapamycin) is a highly conserved Ser/Thr kinase and a central controller of cell growth. Using the crystal structure of the related lipid kinase PI3KCgamma, we built a model of the catalytic region of TOR, from the FAT domain to near the end of the FATC domain. The model reveals that activating mutations in TOR, identified in yeast in a genetic selection for Rheb-independence, correspond to hotspots for oncogenic mutations in PI3KCalpha. The activating mutations are in the catalytic domain (helices kalpha3, kalpha9, kalpha11) and the helical domain of TOR. Docking studies with small molecule inhibitors (PP242, NVP-BEZ235, and Ku-0063794) show that drugs currently in development utilize a novel pharmacophore space to achieve specificity. Thus, our model provides insight on the regulation of TOR and may be useful in the design of new anticancer drugs.

Figure 1

Structural alignment of TORs and PI3KC. The TORs are human TOR (NP_004949.1), S. cerevisiae TOR1 (P35169.3) (TOR1Sc) and TOR2 (P32600.3) (TOR2Sc), and S. pombe TOR2 (NP_595359.2) (TOR2Sp). The PI3KCs are human PI3KCα (CAA82333.1) and pig PI3KCγ (NP_999104.1). The structural elements are drawn from 1e8x and color-coded according to a scheme used throughout for TOR domains (99): FAT, sky blue; FRB, green, CAT, red; FIT (see text for domain definition), cyan; portion of the FATC, magenta. See Methods for tools used in this manual alignment.

Figure 2

TOR compared to PI3KCγ. a) A portion of the FRB domain (99) corresponds to the helical domain (FAT (99) domain), and a portion is the helix kα1 of the catalytic domain of PI3KCγ. Two inserts cannot be modeled to PI3KCγ. One is early in the FIT (99) domain (defined as residues 2427−2516) between the catalytic and FATC domains. The other is in the catalytic domain. Coloring of PI3KCγ (1e8x): blue, helical domain; yellow, catalytic domain. The C-terminal part of the FIT domain forms the C-terminal helices in PI3KCγ (kα10-kα12). A portion of the FATC (99) has no template in 1e8x but either overlaps or is appended to kα12. b). A rotation shows the key structural roles of the RQD helix (kα3) and kα9. The kα9 ends the conserved catalytic domain of TOR. Insertion of the undefined portion of FIT domain is at the two stubs (asterisks).

Figure 3

Residues binding ATP in TOR. a) Orientation of adenine N-6 to Tyr2225 in TOR. Asp2357 is near the γ- and β-phosphates of ATP. b) Lys2187 is near the α-phosphate of ATP.

Figure 4

Pharmacophore space in TOR. a) The γ-phosphate of ATP (gray) is exposed. b) A pharmacophore space lies below the active site. This space is also in the PI3KCγ structure (see Supplementary Figure S1). See ref (99) for definitions of domain acronyms FAT, FRB, CAT, FIT, and FATC.

Figure 5

Location of mutations in model of S. cerevisiae TOR1. a) Mutation of Trp2176 in the adenine pocket increases resistance to caffeine. Mutations of Ile1954 and Ala1957 in the FRB domain activate TOR1. View is from the back. b) Trp2176 is a TOR-specific difference in the adenine pocket. Enlarge to see side chains (orange) of mutated residues.

Figure 6

Sites of activating mutations in S. pombe TOR2. A) Front and B) rear of molecule. Included in view is the site (L2048) of an inactivating mutation of S. pombe TOR2. Double arrow shows the key helices, kα3 (the RQD) and kα9 in PI3K-related kinases. Enlarge to see side chains (orange) of mutated residues.

Figure 7

Manual docking of PP242 in TOR. A) S2 with its methyl group fit in PI3KCγ structure (3ene). B) PP242 hindered by wider isopropyl group in PI3KCγ. C) PP242 can fit into the TOR model. The pyrazolopyrimidine rings of PP242 and S2 were superimposed to position PP242 (see text). The images were generated with MacPyMol (DeLano Scientific).

Figure 8

Optimized binding modes of lead compounds to the model of human TOR defined by AutoDock4 (Scripps). The TOR residues shown all have at least one atom within 4 Å of ligand. Ligands: carbon atoms, gray; nitrogen, blue; oxygen, red. Dashed lines, bonds. A) PP242. Leu2185 is omitted. B) NVP-BEZ235. C) Ku-0063794. Pro2169 and Lys2187 are omitted.

Figure 9

Superposition of the predicted binding modes of PP242, NVP-BEZ235, and Ku-0063794. PP242, carbons, magenta; NVP-BEZ235, carbons, gray; Ku-0063794, carbons, yellow.

Figure 10

Differences in use of a novel pharmacophore space may be responsible for TOR specificity of Ku-0063794. Surface view of TOR generated by MacPymol (Delano Scientific), viewed roughly perpendicular to the space, the surface termed “bottom”. TOR domains are colored as before. A) Docked PP242. B) Docked NVP-BEZ235. C) Docked Ku-0063794. Note how TOR-specific Ku-0063794 packs into and nearly fills this space.