Sandbox Reserved 825

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This Sandbox is Reserved from 06/12/2018, through 30/06/2019 for use in the course "Structural Biology" taught by Bruno Kieffer at the University of Strasbourg, ESBS. This reservation includes Sandbox Reserved 1480 through Sandbox Reserved 1543.
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FKBP12-rapamycin binding domain of mTORFKBP12-rapamycin binding domain of mTOR

FRB domain (2NPU) of mTOR is responsible for the binding of the inhibitory cyclic macrolide Rapamycin

IntroductionIntroduction

Cartoon model of the FRB domain of mTOR:

N               C
Drag the structure with the mouse to rotate

As a member of the phosphatidylinositol 3-kinase-related kinases (PIKK) the mammalian targert of rapamycin (mTOR) is a multi domain protein which is involved in the regulation of cell growth and an important target of survival signals in cancer cells.

The sequence of the 2549 residues is highly conserved across eukaryotes (40-60% sequence identity). The protein consists of several functional domains:
At the N-terminus there are twelve HEAT repeats followed by a central FAT domain (residues 1513-1910), a FRB domain (residues 2015-2114), a serine-threonine kinase domain (residues 2181-2484) and a C-terminal FATC domain (residues 2515-2549).


Fig.1 Structure of mTOR http://dev.biologists.org/content/138/16/3343.full


The FKBP12-rapamycin binding (FRB) domain mediates ligand-dependent regulation of the kinase domain by binding different molecules. FRB binds the inhibitory cyclic macroloide rapamycin in complex with the small peptidyl-prolyl cis-trans isomerase FKBP12 leading to a decreased activity of the kinase domain. Due to its inhibitory effect rapamycin has been a widely used tool for studying mTOR.
FRB is also capable of binding the activator phosphatidic acid and small molecules, for example amino acids like leucine. [1]


Structure of FRBStructure of FRB

Fig.2 A representative view of phosphatidic acid docked into its binding site on the FRB domain.

Surface structure of FRB

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The FRB domain is made up of a disordered N-terminal domain and a four joined by short loops (α-helix1: W2023-G2040, α-helix2: V2044-R2060, α-helix3: L2065-L2090, α-helix4: V2094-S2112). [2] Helix 3 contains a of approximately 45° and its N-terminal half is largely disordered.

The surface structure reveals two main interaction sites, a shallow made up of helix 1 and helix 4 which is responsible for binding rapamycin and a between helix 2 and helix 3. This cleft contains charged and hydrophobic residues and is expected to function as a binding site for small molecules to regulate mTOR activity.

The binding sites for phosphatidic acid and rapamycin show significant (L2031, F2039, Y2105, H2106, R2109) suggesting that rapamycin inhibits kinase activity of mTOR by blocking access of the activator phosphatidic acid.
Active residues are exposed to water and interact with the ligand. Passive residues are also in contact with water and are situated proximate to the active ones.[3]
There are whose side chains are exposed to the solvent and who play an active role in phosphatidic acid binding (E2031, F2039, Y2105, H2106, R2109). Those are the residues that also take part in rapamycin binding. The positive charged arginine residue R2109 plays a key role in the binding of phosphatidic acid as it binds to the negetively charged phosphate group.

at the FRB domain contribute passively to the binding of the activator (L2031, S2035, W2101).

Fig.3 A representative view of the mTOR inhibitor HTS-1 docked into its binding site on the FRB domain on the left. On the right, the location and conformation of rapamycin bound to the FRB domain in the ternary complex formed with FKBP12 is illustrated, with the domain shown in the same orientation as on the left.




Other molecules like the novel class inhibitor HTS-1 (4-[6-{[(1S,2R)-2-(benzyloxy)cyclopentyl]acetyl}-4-(2-thienyl)pyridin-2-yl]-4-oxobutanoic acid) are also capable of inhibiting the kinase activity of mTOR by partially occupying the binding site for phosphatidic acid. There are residues at the FRB domain that are predominantly involved in HTS-1 binding (active residues: E2032, S2035, Y2038, F2039, T2098, W2101, Y2105, F2108, passive residues: H2028, L2031).
At least of those residues also take part in phosphatidic acid binding (L2031, W2101, E2032, F2039, Y2105, S2035). [4]



Biological SignificanceBiological Significance

MTOR has been found to form two distinct complexes called mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2).
While mTORC1 is bound and inhibited by rapamycin-FKBP12 mTORC2 is not affected by rapamycin presence. [5] Depending on the formed complex mTOR activity can provoke different cellular responses. MTORC1 for example leads to activation of translation and inhibition of autophagy.

MTORC2 on the other hand regulates the assembly of cytoskeleton and activates the Protein Kinase B (Akt) which plays a central role in the phosphoinositide 3-kinase pathway , which leads to cell survival and S-phase entry and is revealed to be overactive in many human cancers. [6]

The FRB domain is responsible for the ligand-mediated regulation of mTOR activity. Though the inhibitor rapamycin has been successfully applied as an immunosuppressant it cannot be used to treat cancer since rapamycin has no effect on mTORC2.
Therefore scientists are interessted in discovering new binding sites for inhibitors (like HTS-1) that inhibit mTORC2 and can help to fight cancer. [7]

ReferencesReferences

  1. ↑ Russell, R. C., Fang, C., & Guan, K. L. (2011). An emerging role for TOR signaling in mammalian tissue and stem cell physiology. Development, 138(16), 3343-3356. doi: http://dev.biologists.org/content/138/16/3343.full
  2. ↑ Veverka V, Crabbe T, Bird I, Lennie G, Muskett FW, Taylor RJ, Carr MD. Structural characterization of the interaction of mTOR with phosphatidic acid and a novel class of inhibitor: compelling evidence for a central role of the FRB domain in small molecule-mediated regulation of mTOR. Oncogene. 2008 Jan 24;27(5):585-95. Epub 2007 Aug 6. PMID:17684489
  3. ↑ Roterman-Konieczna, Irena. Identification of ligand binding site and protein-protein interaction area. Vol. 8. Springer, 2013.
  4. ↑ Ballou LM, Lin RZ. Rapamycin and mTOR kinase inhibitors. J Chem Biol. 2008 Nov;1(1-4):27-36. doi: 10.1007/s12154-008-0003-5. Epub 2008 May, 15. PMID:19568796 doi:http://dx.doi.org/10.1007/s12154-008-0003-5
  5. ↑ Dowling RJ, Topisirovic I, Fonseca BD, Sonenberg N. Dissecting the role of mTOR: lessons from mTOR inhibitors. Biochim Biophys Acta. 2010 Mar;1804(3):433-9. doi: 10.1016/j.bbapap.2009.12.001. , Epub 2009 Dec 11. PMID:20005306 doi:http://dx.doi.org/10.1016/j.bbapap.2009.12.001
  6. ↑ Cantley LC. The phosphoinositide 3-kinase pathway. Science. 2002 May 31;296(5573):1655-7. PMID:12040186 doi:http://dx.doi.org/10.1126/science.296.5573.1655
  7. ↑ Ciuffreda L, Di Sanza C, Incani UC, Milella M. The mTOR pathway: a new target in cancer therapy. Curr Cancer Drug Targets. 2010 Aug;10(5):484-95. PMID:20384580

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