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PDBsum entry 1v0d
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Contents |
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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Structural mechanism for inactivation and activation of cad/dff40 in the apoptotic pathway.
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Authors
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E.J.Woo,
Y.G.Kim,
M.S.Kim,
W.D.Han,
S.Shin,
H.Robinson,
S.Y.Park,
B.H.Oh.
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Ref.
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Mol Cell, 2004,
14,
531-539.
[DOI no: ]
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PubMed id
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Abstract
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CAD/DFF40 is responsible for the degradation of chromosomal DNA into nucleosomal
fragments and subsequent chromatin condensation during apoptosis. It exists as
an inactive complex with its inhibitor ICAD/DFF45 in proliferating cells but
becomes activated upon cleavage of ICAD/DFF45 into three domains by caspases in
dying cells. The molecular mechanism underlying the control and activation of
CAD/DFF40 was unknown. Here, the crystal structure of activated CAD/DFF40
reveals that it is a pair of molecular scissors with a deep active-site crevice
that appears ideal for distinguishing internucleosomal DNA from nucleosomal DNA.
Ensuing studies show that ICAD/DFF45 sequesters the nonfunctional CAD/DFF40
monomer and is also able to disassemble the functional CAD/DFF40 dimer. This
capacity requires the involvement of the middle domain of ICAD/DFF45, which by
itself cannot remain bound to CAD/DFF40 due to low binding affinity for the
enzyme. Thus, the consequence of the caspase-cleavage of ICAD/DFF45 is a
self-assembly of CAD/DFF40 into the active dimer.
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Figure 1.
Figure 1. Structural Aspects of CAD(A) The monomeric
structure. The secondary structures in Domains C2 and C3 are
numbered in the order of appearance in the primary sequence. The
invisible Domain C1 is represented as a sphere. The
catalytically important histidine and lysine residues are shown
in ball-and-sticks. The Zn^2+ binding site is shown with the
Zn^2+ in orange and the cysteinyl sulfur in yellow. The inset
shows the detailed interactions of the catalytic residues
together with the final 2F[o] − F[c] electron density map
(2.6 Å, 1.0 σ). The putative Mg^2+ is represented as a
yellow sphere. Asp262, His308, and two loosely bound water
molecules provide the metal coordination arms. Asn260 and a
water molecule are on the hydrogen-bonded network with a
metal-coordinating water.(B) The dimeric structure. In the side
view (top), one subunit in blue is oriented similar to that in
(A). The catalytic residues and the Zn^2+ binding site are
shown. A DNA strand shows CAD on the same scale. The top view
(bottom) is looking down along the molecular 2-fold axis. The
crevice between the C3 domains spans 14 base pairs of the
modeled DNA. Of note, the longest helix α4 fits into the major
groove of the DNA.
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Figure 4.
Figure 4. Estimation of Molecular WeightThe apparent
molecular weight of each indicated complex was analyzed with a
HiLoad 26/60 Superdex 75 column. The proteins were eluted at a
rate of 1.5 ml/min with 30 mM TrisHCl buffer (pH 8.0) containing
100 mM NaCl and 3 mM dithiothreitol. For clarity, the elution
profiles for only CAD:ICAD and CAD are shown. The size marker
proteins were the TRAIL:DR5 complex (99 kDa), albumin (66 kDa),
ovalbumin (45 kDa), chymotrypsinogen (25 kDa), and ribonuclease
A (14 kDa). The calculated molecular weights of CAD, ICAD-L,
ICAD-S, I1, I1-I2, and I2-I3 that we generated are 37.53, 34.42,
28.77, 12.87, 24.76, and 21.56 kDa, respectively.
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The above figures are
reprinted
by permission from Cell Press:
Mol Cell
(2004,
14,
531-539)
copyright 2004.
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