PDBsum entry 1v0d

Hydrolase

PDB id: 1v0d

Contents

Protein chain

245 a.a. *

Metals

_PB

_ZN

_MG

Waters ×16

* Residue conservation analysis

PDB id:

1v0d

Name:

Hydrolase

Title:

Crystal structure of caspase-activated dnase (cad)

Structure:

DNA fragmentation factor 40 kda subunit. Chain: a. Fragment: residues 1-329. Synonym: dff-40, caspase-activated deoxyribonuclease, caspase- activated dnasecad. Engineered: yes

Source:

Mus musculus. Mouse. Organism_taxid: 10090. Expressed in: escherichia coli. Expression_system_taxid: 469008.

Biol. unit:

Dimer (from PDB file)

Resolution:

2.60Å R-factor: 0.218 R-free: 0.246

Authors:

E.-J.Woo,Y.-G.Kim,M.-S.Kim,W.-D.Han,S.Shin,B.-H.Oh

Key ref:

E.J.Woo et al. (2004). Structural mechanism for inactivation and activation of CAD/DFF40 in the apoptotic pathway. Mol Cell, 14, 531-539. PubMed id: 15149602 DOI: 10.1016/S1097-2765(04)00258-8

Date:

26-Mar-04 Release date: 21-May-04

Protein chain

O54788  (DFFB_MOUSE) -  DNA fragmentation factor subunit beta from Mus musculus

Seq: 344 a.a.

Struc: 245 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.3.-.-.-

DOI no: 10.1016/S1097-2765(04)00258-8

Mol Cell 14:531-539 (2004)

PubMed id: 15149602

Structural mechanism for inactivation and activation of CAD/DFF40 in the apoptotic pathway.

E.J.Woo, Y.G.Kim, M.S.Kim, W.D.Han, S.Shin, H.Robinson, S.Y.Park, B.H.Oh.

ABSTRACT

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.

Selected figure(s)

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.

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.

The above figures are reprinted by permission from Cell Press: Mol Cell (2004, 14, 531-539) copyright 2004.

Figures were selected by an automated process.