mTORΔN-mLST8 (4JSN) from Homo sapiens
Created by: Michael Pierce
mTORΔN-mLST8 (PDB ID=4JSN) is a complex of the proteins mTOR and mLST8, a subunit of mTOR. mTOR, also known as "mammalian target of rapamycin", from Homo sapiens belongs to the phosphoinositide 3-kinase (PI3K)-related protein kinase (PIKK) family. It is a slightly acidic 340360 Da protein with an isoelectric point of 6.39, composed primarily of α-helices (1). The PIKK family is a class of proteins that is involved in cell proliferation, apoptosis, cell differentiation, and embryonic development (2). mTOR plays a central role in regulating cellular growth and metabolism as well as survival in response to extracellular factors, including nutrients, growth factors, hormones, and stress signals. mTOR has been shown to be ubiquitous to many mammals, as demonstrated by PSI-Blast results (3). When deregulated, mTOR is involved cancer metastasis, making research of the protein important in the field of oncology (4,5). mLST8 interacts directly with mTOR, increasing kinase activity and stabilizing key mTOR interactions in nutrient-poor environments (6).
mTOR is composed of two distinct complexes, mTORC1 and mTORC2. Crystallized mTORΔN-mLST8 is comparable to mTORC1 (2). mTORΔN-mLST8 has three important domains: the kinase domain (KD), the FAT domain, and the mLST8 domain. The immunosuppressant drug rapamycin is known to inhibit mTOR's function (7).
The KD consists oftwo lobes designated N and C. The N-lobe, located adjacent to the FRB domain, discussed later, contains the functionally integral kα1 helix. This helix packs in the surface of a conserved β-sheet also located in the N-lobe of the KD. The C-lobe is located in close vicinity to the catalytic cleft and contains four important structural insertions, LBE (which importantly binds mLST8), kαAL, kα9B, and FATC. Together these insertions form a structural spine around the activation loop, a ~30 residue segment that plays a large role in function and regulation of kinase activity. This role includes providing an active-site residue and undergoing a conformational change upon kinase activation. The C-terminal half of the FATC insertion forms three helices that interact with the activation loop, and one helix that interacts with the LBE insertion. The FATC-activation loop and LBE-FATC interactions are thought to stabilize the activation loop directly and indirectly, respectively. The 11-residue kα9b insertion acts to plug one end of the catalytic cleft, importantly restricting access to the substrate-binding site (2).
The active site of the KD contains multiple conserved residues necessary for regulation. C-lobe Asn-2343 and Asp-2357 both serve as metal ligand binding sites, and Asp-2338 orients and activates the substrate hydroxyl group for nucleophilic attack. His 2340 plays an important role in kinase activity, demonstrated by the lack of activity when the residue is mutated (2).
The FRB, or FKBP12-rapamycin-binding domain, is located within the KD N-lobe and acts as a gatekeeper to the substrate-binding site. This domain extends the N-lobe side of the KD, providing the cleft with a deep V shape, which, along with the kα9b insertion, restricts access to the substrate-binding site. Within the FRB, the rapamycin-binding site is located in close proximity to the protein’s active site. When rapamycin-FKBP12 binds to the FRB, FKBP12 is located only 8Å from the mLST8 domain of mTOR, effectively blocking all access to the substrate-binding site (2). Because of this regulatory action, rapamycin is being carefully researched as an anti-cancer drug (8). Along with aiding in restriction to the active site, FRB also facilitates S6K1 (a ribosomal protein kinase) access to the active site, yielding increased kinase activity. The FRB importantly contains a conserved core residue, Trp-2027, which, if mutated, results in the abolishment of kinase activity (2).
The FAT domain (named after FRAP, ATM, and TRRAP) of mTORΔN-mLST8 consists of 28 α-helices arranged in α-α-helix repeats. Helices α1-α22 form three domains (TRD1,TRD2, and TRD3), and helices α23-α28 form one domain (HRD). Together the domains form a C-shaped clamp that surrounds the KD, facilitating conserved interactions between the KD and both TRD1 and HRD. These interactions are conserved among the PIKK family, and are therefore assumed to be important stabilizing features. The TRD1 interaction with the C-lobe side of the KD includes a conserved hydrogen bond between Glu-1401/Arg-2317. The HRD interaction with both the N-lobe and C-lobe sides of the KD includes conserved hydrogen bonds between Arg-1905/Glu-2419, and Gln-1941/Gln-2200. The HRD interaction also involves a salt bridge between HRD and kα9, specifically between Glu-2419/Arg-1905, elimination of which results in a loosening of the framework that restricts access to the active site (2).
The mLST8 domain is composed of 7 WD40 (a circular motif of approximately 40 amino acids) repeats. At one end of this motif is a surface that extends to the LBE region of the C-lobe KD (9). The interaction between mLST8 and LBE involves mostly polar residues of LBE and polar or aromatic residues of mLST8, and contains a large number of hydrogen bonds. This interaction stabilizes LBE and influences the LBE/FATC/activation-loop interaction spine discussed in Par 2, supporting the idea that mLST8 is a necessary subunit of mTOR activation.
The tertiary structure of the mTORΔN-mLST8 catalytic KD is shown to be similar to that of the crystallized PI3K-γ in complex with AMG511 (PDB ID=4FLH) (10). PI3K-γ is a member of the IB class of PI3K kinases, which are functionally very similar to mTOR, also acting as Serine/Threonine kinases. Like that of mTORΔN-mLST8, the catalytic KD of PI3K-γ is composed of N and C lobes (11). The regions of greatest similarity between the two proteins are from kβ3 to kα3 of the N-lobe and from the beginning of the C-lobe to kβ10. These regions are semi-conserved in most protein kinases. This is likely due to the necessary redundancy of protein kinases. The C-lobe contains a segment very similar to the activation loop of mTOR, located from AA 964-988, which is crucial in the specificity of PI3Ks. The substrate-binding domain of PI3Kγ is composed primarily of β-sheets, compared to the FRB domain of mTOR, which is composed primarily of α-helices (2).
Multiple substrates inhibit the ATP-binding capacity of mTOR, eliminating or decreasing kinase activity. The first of these is Torin2, a highly specific ATP-inhibitor of mTOR. Torin2 contains a tricyclic benzoapthyridinone ring that binds to the adenine site and forms a hydrogen bond to the ‘hinge’ between mTOR’s N and C KD lobes. This bond mimics one of the bonds formed when ATP, as well as other inhibitors, bind. The aminopyridine group of Torin2 extends to the hydrophobic pocket located deep within the catalytic cleft of mTOR, another feature of other inhibitors. Unique to Torin2, however, is the stacking of a tricyclic benzoapthyridinone ring with Trp 2239. When the comparable bond forms with ATP, only 3 atoms of ATP are involved, but when Torin2 binds, a ten-atom portion is involved. This fact helps explain the high potency of the inhibitor. A second ATP-inhibitor is PP242, which alters the conformation of mTOR when bound. The catalytic cleft’s hydrophobic pocket becomes deeper, allowing the hydroxyindole group of PP242 to reach into the new space. This conformational change involves the side chain ofTyr 2225, which swings to reveal the deeper pocket. This change necessitates Leu 2354 to swing out of the way of Tyr. Also involved is the Gln 2223 side chain, which plugs the gap left by the conformation. Leu 2354 is not present in PI3Ks, explaining PP242’s specificity to mTOR.
Together the domains of mTORΔN-mLST8 act in an intrinsically active manner. The secondary structural arrangements of these domains are important when explaining stability and function. Though mTOR is similar in structure and function to other protein kinases, after examining the structure carefully it is apparent that the protein is highly specific, in more ways than one.