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Characterization of the immersion properties of the peripheral membrane anchor of the FATC domain of the kinase "Target of rapamycin" by NMR, oriented CD spectroscopy, and MD simulations.
J. Phys. Chem. B 118, 4817-4831 (2014)
The multidomain ser/thr kinase "target of rapamycin" (TOR) centrally controls eukaryotic growth and metabolism. The C-terminal FATC domain is important for TOR regulation and was suggested to directly mediate TOR-membrane interactions. Here, we present a detailed characterization of the membrane immersion properties of the oxidized and reduced yeast TOR1 FATC domain (2438-2470 = y1fatc). The immersion depth was characterized by NMR-monitored interaction studies with DPC micelles containing paramagnetically tagged 5-or 16-doxyl stearic acid (5-/16-SASL) and by analyzing the paramagnetic relaxation enhancement (PRE) from Mn2+ in the solvent. Complementary MD-simulations of micellar systems in the absence and presence of protein showed that 5-/16-SASL can move in the micelle and that 16-SASL can bend such that the doxyl group is close to the headgroup region and not deep in the interior as commonly assumed. Based on oriented CD (OCD) data, the single α-helix of oxidized/reduced y1fatc has an angle to the membrane normal of ∼30-60°/ ∼ 35-65°in neutral and ∼5-35°/∼0-30°in negatively charged bilayers. The presented experimentally well-founded models help to better understand how this redox-sensitive peripheral membrane anchor may be part of a network of protein-protein and protein-membrane interactions regulating TOR localization at different cellular membranes. Moreover, the presented work provides a good methodological reference for the structural characterization of other peripherally membrane associating proteins.
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Publication type
Article: Journal article
Document type
Scientific Article
Keywords
Nuclear-magnetic-resonance; Micelle-bound Peptides; Particle Mesh Ewald; Circular-dichroism; Molecular-dynamics; Relaxation Enhancements; 3-dimensional Structure; Terminal Region; Lipid-bilayers; Growth-control
ISSN (print) / ISBN
1520-6106
e-ISSN
1520-5207
Journal
Journal of Physical Chemistry B
Quellenangaben
Volume: 118,
Issue: 18,
Pages: 4817-4831
Publisher
American Chemical Society (ACS)
Publishing Place
Washington
Non-patent literature
Publications
Reviewing status
Peer reviewed
Institute(s)
Institute of Structural Biology (STB)