期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 285, 期 9, 页码 6681-6692出版社
ELSEVIER
DOI: 10.1074/jbc.M109.019968
关键词
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资金
- National Institutes of Health [GM060001, P50 CA094056]
- National Science Foundation Division of Biological Infrastructure for Peptide Synthesis Instrumentation [0500580]
- Direct For Biological Sciences
- Div Of Biological Infrastructure [0500580] Funding Source: National Science Foundation
The Notch signaling pathway is a cell-cell communication network giving rise to cell differentiation during metazoan development. Activation of the pathway releases the intracellular portion of the Notch receptor to translocate to the nucleus, where it is able to interact with the effector transcription factor CSL, converting CSL from a transcriptional repressor to an activator. This conversion is dependent upon the high affinity binding of the RAM region of the Notch receptor to the beta-trefoil domain (BTD) of CSL. Here we probe the energetics of binding to BTD of each conserved residue of RAM through the use of isothermal titration calorimetry and single residue substitution. We find that although the highly conserved Phi W Phi P motif is the largest determinant of binding, energetically significant interactions are contributed by N-terminal residues, including a conserved Arg/Lys-rich region. Additionally, we present a thermodynamic analysis of the interaction between the Epstein-Barr virus protein EBNA2 with BTD and explore the extent to which the EBNA2- and RAM-binding sites on BTD are nonoverlapping, as proposed by Fuchs et al. ( Fuchs, K. P., Bommer, G., Dumont, E., Christoph, B., Vidal, M., Kremmer, E., and Kempkes, B. (2001) Eur. J. Biochem. 268, 4639-4646). Combining these results with displacement isothermal titration calorimetry, we propose a mechanism by which the Phi W Phi P motif of RAM and EBNA2 compete with one another for binding at the hydrophobic pocket of BTD using overlapping but specific interactions that are unique to each BTD ligand.
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