Abstract
The Notch signaling pathway plays a key role in a myriad of cellular processes, including cell fate determination. Despite extensive study of the downstream consequences of receptor activation, very little molecular data are available for the initial binding event between the Notch receptor and its ligands. In this study, we have expressed and purified a natively folded wild-type epidermal growth factor-like domain (EGF) 11-14 construct from human Notch-1 and have used flow cytometry and surface plasmon resonance analysis to demonstrate a calcium-dependent interaction with the human ligand Delta-like-1. Site-directed mutagenesis of three of the calcium-binding sites within the Notch-(11-14) fragment indicated that only loss of calcium binding to EGF12, and not EGF11 or EGF13, abrogates ligand binding. Further mapping of the ligand-binding site within this region by limited proteolysis of Notch wild-type and mutant fragments suggested that EGF12 rather than EGF11 contains the major Delta-like-1-binding site. Analysis of an extended fragment EGF-(10-14), where EGF11 is placed in a native context, surprisingly demonstrated a reduction in ligand binding, suggesting that EGF10 modulates binding by limiting access of ligand. This inhibition could be overcome by the introduction of a calcium binding mutation in EGF11, which decouples the EGF-(10-11) module interface. This study therefore demonstrates that long range calcium-dependent structural perturbations can influence the affinity of Notch for its ligand, in the absence of any post-translational modifications.
Highlights
Notch signaling via the classical pathway requires cell-surface expression of a heterodimeric transmembrane form of Notch produced by furin cleavage of the primary translation product in the trans-Golgi network [6, 7]
Following site-directed mutagenesis of three of the calcium-binding sites in Notch epidermal growth factorlike (EGF)-(11–14), we use our binding assays and the subsequent analysis of each mutant protein by limited proteolysis to demonstrate the crucial importance of EGF12 for binding to human Delta-like-1 (hDll-1), allowing further refinement of the ligand-binding region
We demonstrate that Notch EGF10 has a modulatory role on the ability of this region to bind ligand, suggesting that regulation of both this module’s interface with EGF11 and the calcium affinity of EGF11 may play a key role in controlling Notch function
Summary
Cloning and Expression of Notch Constructs—DNA encoding each wild-type Notch fragment was amplified by PCR from Tan (human Notch-1) cDNA and cloned into the prokaryotic expression vectors pQE30 (Qiagen) and pQE30-BirA (a modified version of pQE30 containing nucleotides encoding the recognition sequence of the site-specific biotinylation enzyme BirA [19]). Comparison of wild-type and mutant digest products obtained in the presence of EGTA confirmed that the introduced amino acid substitutions had not had further reaching consequences than the intended abrogation of calcium binding in a single cbEGF domain. Pelleted beads were resuspended in 50 l of HBSS/BSA, and 1 g of biotinylated Notch protein was added prior to incubation on ice for 1 h. Coupled beads were washed with 100 l of HBSS/BSA, resuspended in 50 l of HBSS, 10% fetal calf serum, and sonicated at 20% power for 1 min (Heat Systems, Sonicator) prior to addition to cells. To demonstrate calcium dependence of the Notch-ligand interaction, either 5 mM EGTA was added to the cell/bead binding solution prior to incubation or cell/beads were incubated in HBSS containing 1.26 mM calcium chloride. One-dimensional NMR data were collected with a spectral width of 5494.51 Hz, 4096 complex points, and 512 acquisitions at 25 °C
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