PMID- 18296446
OWN - NLM
STAT- MEDLINE
DCOM- 20080619
LR  - 20220129
IS  - 0021-9258 (Print)
IS  - 0021-9258 (Linking)
VI  - 283
IP  - 17
DP  - 2008 Apr 25
TI  - Localization of the delta-like-1-binding site in human Notch-1 and its modulation 
      by calcium affinity.
PG  - 11785-93
LID - 10.1074/jbc.M708424200 [doi]
AB  - 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.
FAU - Cordle, Jemima
AU  - Cordle J
AD  - Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 
      3QU, United Kingdom.
FAU - Redfieldz, Christina
AU  - Redfieldz C
FAU - Stacey, Martin
AU  - Stacey M
FAU - van der Merwe, P Anton
AU  - van der Merwe PA
FAU - Willis, Antony C
AU  - Willis AC
FAU - Champion, Brian R
AU  - Champion BR
FAU - Hambleton, Sophie
AU  - Hambleton S
FAU - Handford, Penny A
AU  - Handford PA
LA  - eng
GR  - 079440/WT_/Wellcome Trust/United Kingdom
GR  - G9722488/MRC_/Medical Research Council/United Kingdom
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20080222
PL  - United States
TA  - J Biol Chem
JT  - The Journal of biological chemistry
JID - 2985121R
RN  - 0 (Ligands)
RN  - 0 (NOTCH1 protein, human)
RN  - 0 (Receptor, Notch1)
RN  - 0 (Receptors, Notch)
RN  - SY7Q814VUP (Calcium)
SB  - IM
MH  - Animals
MH  - Binding Sites
MH  - CHO Cells
MH  - Calcium/*metabolism
MH  - Cloning, Molecular
MH  - Cricetinae
MH  - Cricetulus
MH  - Humans
MH  - Kinetics
MH  - Ligands
MH  - Magnetic Resonance Spectroscopy
MH  - Protein Conformation
MH  - Receptor, Notch1/chemistry/*physiology
MH  - Receptors, Notch/metabolism
MH  - Surface Plasmon Resonance
EDAT- 2008/02/26 09:00
MHDA- 2008/06/20 09:00
CRDT- 2008/02/26 09:00
PHST- 2008/02/26 09:00 [pubmed]
PHST- 2008/06/20 09:00 [medline]
PHST- 2008/02/26 09:00 [entrez]
AID - S0021-9258(20)61973-1 [pii]
AID - 10.1074/jbc.M708424200 [doi]
PST - ppublish
SO  - J Biol Chem. 2008 Apr 25;283(17):11785-93. doi: 10.1074/jbc.M708424200. Epub 2008 
      Feb 22.