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PDBsum entry 3a8r
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Calcium binding protein
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PDB id
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3a8r
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References listed in PDB file
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Key reference
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Title
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Structure of the n-Terminal regulatory domain of a plant NADPH oxidase and its functional implications.
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Authors
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T.Oda,
H.Hashimoto,
N.Kuwabara,
S.Akashi,
K.Hayashi,
C.Kojima,
H.L.Wong,
T.Kawasaki,
K.Shimamoto,
M.Sato,
T.Shimizu.
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Ref.
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J Biol Chem, 2010,
285,
1435-1445.
[DOI no: ]
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PubMed id
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Note In the PDB file this reference is
annotated as "TO BE PUBLISHED".
The citation details given above were identified by an automated
search of PubMed on title and author
names, giving a
perfect match.
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Abstract
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Plant NADPH oxidases (Rboh; for Respiratory Burst Oxidase Homolog) produce
reactive oxygen species (ROS) that are key regulators of various cellular events
including plant innate immunity. Rbohs possess a highly conserved cytoplasmic
N-terminal region containing two EF-hand motifs which regulates Rboh activity.
Rice (Oryza sativa) RbohB (OsRbohB) is regulated by the direct binding of a
small GTPase (Rac1) to this regulatory region as well as by Ca2+ binding to the
EF-hands. Here, we present the atomic structure of the N-terminal region of
OsRbohB. The structure reveals that OsRbohB forms a unique dimer stabilized by
swapping of EF-hand motifs. We identified two additional EF-hand-like motifs
that were not predicted from sequence data so far. These EF-hand-like motifs
together with the swapped EF-hands form a structure similar to that found in
calcineurin B. We observed conformational changes mediated by Ca2+-binding to
the only one EF-hand. Structure-based in vitro pull-down assays and NMR
titration experiments defined the OsRac1 binding interface within the
coiled-coil region created by the swapping of the EF-hands. In addition, we
demonstrate a direct intramolecular interaction between the N- and the
C-terminus, and that the complete N-terminal cytoplasmic region is required for
this interaction. The structural features and intramolecular interactions
characterized here might be common elements shared by Rbohs that contribute to
the regulation of ROS production.
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Figure 2.
EF-hand motifs of OsRbohB-(138–313) and similar proteins.
A, representation of EF-hand motifs (EF-1, EF-2, EF-like 1, and
EF-like 2). Difference Fourier maps showing contour levels
higher than 5 σ. Ca^2+ ion and water molecules are represented
as a yellow and red spheres, respectively. Amino acid residues
of EF-hand motifs at positions X to Z are shown as sticks. B,
magnified view of EF-hand pairs composed of EF-1 and EF-2, and
EF-like 1 and EF-like 2, respectively. Residues forming the
hydrophobic cores are shown as white sticks. C, hydrophobic
pockets of OsRbohB-(138–313), calcineurin B, and recoverin.
Residues forming the hydrophobic pocket are shown as white
sticks. The pocket of OsRbohB-(138–313) formed by swapped
EF-hands and EF-hand-like motifs is occupied by an N-terminal
helix (blue) protruding from the core domain. The pockets of
calcineurin B and recoverin are occupied by α-helices
protruding from calcineurin A and the C-terminal region (blue),
respectively.
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Figure 4.
Interaction between OsRbohB-(138–313) and OsRac1. In vitro
pulldown assays using OsRbohB-(138–313) mutants and
GST-OsRac1. Mutation and deletion sites are mapped onto the
structure of OsRbohB-(138–313) shown as a ribbon model and as
surface representations (two views from opposite sides).
Residues that are necessary to maintain the full binding
affinity are colored magenta, whereas residues exhibiting no or
little effect on the binding are shown in blue.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2010,
285,
1435-1445)
copyright 2010.
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