Characterization of the Immersion Properties of the Peripheral Membrane Anchor of the FATC Domain of the Kinase 鈥淭arget of Rapamycin鈥?by NMR, Oriented CD Spectroscopy, and MD Simulations

详细信息    查看全文

• 作者：Lisa A. M. Sommer ; J. Joel Janke ; W. F. Drew Bennett ; Jochen B眉rck ; Anne S. Ulrich ; D. Peter Tieleman ; Sonja A. Dames
• 刊名：Journal of Physical Chemistry B
• 出版年：2014
• 出版时间：May 8, 2014
• 年：2014
• 卷：118
• 期：18
• 页码：4817-4831
• 全文大小：896K
• 年卷期：v.118,no.18(May 8, 2014)
• ISSN：1520-5207

文摘

The multidomain ser/thr kinase 鈥渢arget of rapamycin鈥?(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鈥?470 = 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 伪-helix of oxidized/reduced y1fatc has an angle to the membrane normal of 30鈥?0掳/ 35鈥?5掳 in neutral and 5鈥?5掳/0鈥?0掳 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鈥損rotein and protein鈥搈embrane 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.