Abstract
The target of rapamycin (TOR) is a conserved eukaryotic Ser/Thr kinase that regulates cellular growth in response to the nutrient and energy state. TOR signaling plays an important role in the development of diseases such as cancer, obesity, and diabetes and in different redox-sensitive processes (hypoxia, apoptosis, and aging). Because TOR has been detected at different cellular membranes and in the nucleus, its localization may influence the specific signaling readout. To better understand how TOR can associate with different membranes, the lipid-binding properties of the redox-sensitive yeast TOR1 FATC domain (y1fatc) have been characterized by solution NMR spectroscopy. Binding studies with different lipids indicate that y1fatc interacts specifically with a membrane-mimetic environment but appears not to recognize a specific lipid headgroup. In both, the structures of oxidized and reduced micelle-bound y1fatc, residues Ile-2456 to Trp-2470 of the lipid-binding motif form a hydrophobic bulb that has a rim of charged residues. The diffusion constants for both micelle-bound states are consistent with the rotational correlation times from the analysis of the (15)N relaxation data. Based on the K(d) values, the oxidized form (K(d) approximately 0.31 mm) binds dodecyl phosphocholine micelles slightly tighter than the reduced form (K(d) approximately 1.86 mM). Binding studies with y1fatc in which one or both tryptophans (Trp-2466 and Trp-2470) were replaced by alanine suggest that these residues are important for the exact positioning in the membrane and that the other aromatic (His-2462, Tyr-2463, and Phe-2469) and aliphatic residues (Ile-2456, Leu-2459, Ile-2464, and Pro-2468) in the lipid-binding motif contribute significantly to the affinity.
Highlights
target of rapamycin (TOR) proteins have a molecular mass of ϳ280 kDa and share a modular structure (Fig. 1)
To better understand how TOR can associate with different membranes, the lipidbinding properties of the redox-sensitive yeast TOR1 FATC domain (y1fatc) have been characterized by solution NMR spectroscopy
For the structure determination of reduced y1fatc bound to dodecyl phosphocholine (DPC) micelles, the protein was first reduced with 20 mM tris(2-carboxyethyl)phosphine for ϳ10 –30 min, before 170 mM d38-DPC were added
Summary
Sample Preparation—The FATC domain of yeast TOR1 (2438 –2470, y1fatc) was expressed and purified as described previously [15]. 1,2-. For the structure determination of oxidized y1fatc bound to DPC micelles, samples contained either 190 –220 mM d38-DPC or 140 mM d38-DPC/10 mM DPC. For the structure determination of reduced y1fatc bound to DPC micelles, the protein was first reduced with 20 mM tris(2-carboxyethyl)phosphine for ϳ10 –30 min, before 170 mM d38-DPC were added. The protein concentration was 0.063 mM for oxidized and 0.409 mM for reduced y1fatc with 170 mM DPC; 0.051 mM for both with 30 mM DPC; 0.409. Kd Determination—The dissociation constant for oxidized and reduced y1fatc bound to DPC micelles was determined using an approach similar to the one described in previous studies [33, 34]. To calculate the Kd for the protein-micelle complex, fb,p was derived from the measured diffusion constants as described above [33, 34]. Backbone dihedral angle restraints for and were derived based on the determined 13C␣ chemical shifts and on initial structure calculations
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