PDBsum entry 2kit

Transferase

PDB id: 2kit

Contents

Protein chain

33 a.a.

References listed in PDB file

Key reference

Title

Structural basis for the association of the redox-Sensitive target of rapamycin fatc domain with membrane-Mimetic micelles.

Author

S.A.Dames.

Ref.

J Biol Chem, 2010, 285, 7766-7775. [DOI no: 10.1074/jbc.M109.058404]

PubMed id

20042596

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.

Figure 4.
The NMR structures of DPC-micelle bound oxidized (A) and reduced (B) y1fatc. Both plots show a ribbon representation of the superposition of the 20 lowest energy structures to the left and a line representation to the right. The side chains of the disulfide bonding forming cysteines (Cys-2460 and Cys-2467) are colored yellow. In the line representation the backbone of the α-helical stretch is depicted in red. Aromatic side chains are shown in light (F, H, Y) and dark (W) green, hydrophilic side chains in light blue, and those containing methyl groups in dark blue. Prolines are colored purple, and the conserved glycine (Gly-2465) that facilitates the chain reversal is in orange. C shows a superposition of the membrane-binding region of reduced (green, cysteine side chains in red) and oxidized (blue, cysteine side chains in yellow) y1fatc bound to DPC micelles. The side chains of Arg-2458 and Gln-2461 have been omitted for clarity. All structure images were made with the programs MolMol (55) and POV-Ray.

Figure 5.
Model of y1fatc membrane association. A, for this representation, the results from the titration with DPC (Fig. 2) have been mapped onto a space-filled model of the structure of oxidized y1fatc bound to DPC micelles; the color coding is the same as in Fig. 2B. B, the top shows the lowest energy structure of micelle-bound oxidized and reduced y1fatc with the same color coding as in Fig. 4, A and B, and with the bonds between heavy atoms shown in a neon representation. For the side chains of Trp-2466 and Phe-2469 of micelle-bound oxidized y1fatc several conformers are shown to represent the structural heterogeneity observed in the ensemble (Fig. 4A). Below, the surface charge distribution of both structures was visualized. Positively charged areas are colored blue and negatively charged ones are red. DPC with its negatively charged phosphate and its positively charged choline group is schematically depicted to the left. The solvent-membrane interface that has been estimated based on the titrations with DPC and the distribution of surface charges is indicated by a dotted black line. Views of A and B, where the structures have been rotated by 180° around the vertical axes are given in supplemental Fig. 10, A and B. All structure images were made with the programs MolMol (55) and POV-Ray. Supplemental Fig. 10C shows further structure images of a DPC micelle (56) and oxidized micelle-bound y1fatc at the same scale.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2010, 285, 7766-7775) copyright 2010.