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J. Biol. Chem., Vol. 283, Issue 17, 11785-11793, April 25, 2008

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Localization of the Delta-like-1-binding Site in Human Notch-1 and Its Modulation by Calcium Affinity^*

Jemima Cordle¹, Christina RedfieldZ², Martin Stacey, P. Anton van der Merwe³, Antony C. Willis^¶, Brian R. Champion^||, Sophie Hambleton^**, and Penny A. Handford²⁴

From the Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, ^¶Medical Research Council Immunochemistry Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, ^||Celldex Therapeutics Ltd., Cambridge Science Park, Cambridge CB4 0PE, and ^**Medical Research Council Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford OX3 9DS, United Kingdom

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.

Received for publication, October 10, 2007 , and in revised form, January 4, 2008.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table 1 and Figs. 1-4.

¹ Supported by a Medical Research Council studentship.

² Supported by the Wellcome Trust.

³ Supported by the Medical Research Council.

⁴ Supported by the Medical Research Council. To whom correspondence should be addressed. Tel.: 44-1865-285347; Fax: 44-1865-285327; E-mail: penny.handford{at}bioch.ox.ac.uk.

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