5CZX

Crystal structure of Notch3 NRR in complex with 20358 Fab


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.10 Å
  • R-Value Free: 0.226 
  • R-Value Work: 0.190 

wwPDB Validation   3D Report Full Report


Ligand Structure Quality Assessment 


This is version 1.3 of the entry. See complete history


Literature

Characterization of activating mutations of NOTCH3 in T-cell acute lymphoblastic leukemia and anti-leukemic activity of NOTCH3 inhibitory antibodies.

Bernasconi-Elias, P.Hu, T.Jenkins, D.Firestone, B.Gans, S.Kurth, E.Capodieci, P.Deplazes-Lauber, J.Petropoulos, K.Thiel, P.Ponsel, D.Hee Choi, S.LeMotte, P.London, A.Goetcshkes, M.Nolin, E.Jones, M.D.Slocum, K.Kluk, M.J.Weinstock, D.M.Christodoulou, A.Weinberg, O.Jaehrling, J.Ettenberg, S.A.Buckler, A.Blacklow, S.C.Aster, J.C.Fryer, C.J.

(2016) Oncogene 35: 6077-6086

  • DOI: https://doi.org/10.1038/onc.2016.133
  • Primary Citation of Related Structures:  
    5CZV, 5CZX

  • PubMed Abstract: 

    Notch receptors have been implicated as oncogenic drivers in several cancers, the most notable example being NOTCH1 in T-cell acute lymphoblastic leukemia (T-ALL). To characterize the role of activated NOTCH3 in cancer, we generated an antibody that detects the neo-epitope created upon gamma-secretase cleavage of NOTCH3 to release its intracellular domain (ICD3), and sequenced the negative regulatory region (NRR) and PEST (proline, glutamate, serine, threonine) domain coding regions of NOTCH3 in a panel of cell lines. We also characterize NOTCH3 tumor-associated mutations that result in activation of signaling and report new inhibitory antibodies. We determined the structural basis for receptor inhibition by obtaining the first co-crystal structure of a NOTCH3 antibody with the NRR protein and defined two distinct epitopes for NRR antibodies. The antibodies exhibit potent anti-leukemic activity in cell lines and tumor xenografts harboring NOTCH3 activating mutations. Screening of primary T-ALL samples reveals that 2 of 40 tumors examined show active NOTCH3 signaling. We also identified evidence of NOTCH3 activation in 12 of 24 patient-derived orthotopic xenograft models, 2 of which exhibit activation of NOTCH3 without activation of NOTCH1. Our studies provide additional insights into NOTCH3 activation and offer a path forward for identification of cancers that are likely to respond to therapy with NOTCH3 selective inhibitory antibodies.


  • Organizational Affiliation

    Developmental and Molecular Pathways, Novartis Institutes for Biomedical Research, Cambridge, MA, USA.


Macromolecules
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Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Neurogenic locus notch homolog protein 3
A, B
271Homo sapiensMutation(s): 0 
Gene Names: NOTCH3
UniProt & NIH Common Fund Data Resources
Find proteins for Q9UM47 (Homo sapiens)
Explore Q9UM47 
Go to UniProtKB:  Q9UM47
PHAROS:  Q9UM47
GTEx:  ENSG00000074181 
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupQ9UM47
Sequence Annotations
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  • Reference Sequence
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Entity ID: 2
MoleculeChains Sequence LengthOrganismDetailsImage
20358 Fab heavy chainC,
E [auth H]
226Homo sapiensMutation(s): 0 
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
Sequence Annotations
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  • Reference Sequence
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 3
MoleculeChains Sequence LengthOrganismDetailsImage
20358 Fab light chainD,
F [auth L]
214Homo sapiensMutation(s): 0 
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
Sequence Annotations
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  • Reference Sequence
Small Molecules
Ligands 4 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
NAG
Query on NAG

Download Ideal Coordinates CCD File 
G [auth A],
N [auth B]
2-acetamido-2-deoxy-beta-D-glucopyranose
C8 H15 N O6
OVRNDRQMDRJTHS-FMDGEEDCSA-N
EDO
Query on EDO

Download Ideal Coordinates CCD File 
AA [auth L]
K [auth A]
L [auth A]
M [auth A]
R [auth B]
AA [auth L],
K [auth A],
L [auth A],
M [auth A],
R [auth B],
S [auth B],
U [auth C],
V [auth D],
W [auth D],
Y [auth H]
1,2-ETHANEDIOL
C2 H6 O2
LYCAIKOWRPUZTN-UHFFFAOYSA-N
CA
Query on CA

Download Ideal Coordinates CCD File 
H [auth A]
I [auth A]
J [auth A]
O [auth B]
P [auth B]
H [auth A],
I [auth A],
J [auth A],
O [auth B],
P [auth B],
Q [auth B]
CALCIUM ION
Ca
BHPQYMZQTOCNFJ-UHFFFAOYSA-N
CL
Query on CL

Download Ideal Coordinates CCD File 
T [auth C],
X [auth H],
Z [auth L]
CHLORIDE ION
Cl
VEXZGXHMUGYJMC-UHFFFAOYSA-M
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.10 Å
  • R-Value Free: 0.226 
  • R-Value Work: 0.190 
  • Space Group: P 21 21 21
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 88.34α = 90
b = 123.86β = 90
c = 150.57γ = 90
Software Package:
Software NamePurpose
HKL-2000data reduction
SCALEPACKdata scaling
PHENIXphasing
BUSTERrefinement
HKL-2000data scaling

Structure Validation

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Ligand Structure Quality Assessment 


Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2016-06-01
    Type: Initial release
  • Version 1.1: 2016-12-07
    Changes: Database references
  • Version 1.2: 2020-07-29
    Type: Remediation
    Reason: Carbohydrate remediation
    Changes: Data collection, Derived calculations, Structure summary
  • Version 1.3: 2023-09-27
    Changes: Data collection, Database references, Refinement description, Structure summary