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.
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 Mn(2+) 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.
Chair of Biomolecular NMR Spectroscopy, Department of Chemistry, Technische Universität München , Lichtenbergstrasse 4, 85747 Garching, Germany.
Lisa A M
LA
Sommer
J Joel
JJ
Janke
W F Drew
WF
Bennett
Jochen
J
Bürck
Anne S
AS
Ulrich
D Peter
DP
Tieleman
Sonja A
SA
Dames
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D008823:Micelles; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D019869:Phosphatidylinositol 3-Kinases / Q000737:chemistry*; D010767:Phosphorylcholine / Q000031:analogs & derivatives / Q000737:chemistry; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000737:chemistry*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*
D008823:Micelles; D019869:Phosphatidylinositol 3-Kinases; D010767:Phosphorylcholine; D029701:Saccharomyces cerevisiae Proteins; C028810:dodecylphosphocholine; C083324:TOR1 protein, S cerevisiae