1. Volume 14, Issue 4, Pages 531-539 (May 2004)
Structural Mechanism for Inactivation and Activation of CAD/DFF40 in the Apoptotic Pathway 
Eui-Jeon Woo, Yeon-Gil Kim, Min-Sung Kim, Won-Deok Han, Sejeong Shin, Howard Robinson, Sam-Yong Park, Byung-Ha Oh 
Molecular Cell 
Volume 14, Issue 4, Pages (May 2004)
DOI: /S (04)

2. 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 Zn2+ binding site is shown with the Zn2+ in orange and the cysteinyl sulfur in yellow. The inset shows the detailed interactions of the catalytic residues together with the final 2Fo − Fc electron density map (2.6 Å, 1.0 σ). The putative Mg2+ 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 Zn2+ 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.
Molecular Cell  , DOI: ( /S (04) )

3. Figure 2 Zn2+ Binding and Effects of Zinc Removal by o-Phenanthroline
(A) Environment of the Zn2+ binding site. The residues involved in the coordination of Zn2+ (in orange) or intersubunit interactions closer than 4.0 Å are shown in ball-and-sticks. The two subunits of the dimer are indicated by different colors. The hydrogen bond between His242 and Glu251 is the sole intersubunit hydrogen bond.
(B) Inhibitory effect of o-phenanthroline on the DNase activity of CAD. After preincubation of purified activated CAD (200 ng) with o-phenanthroline at the indicated concentration for 15 min at 25°C, 200 ng of plasmid DNA was added and incubated for 30 min at 37°C. The analysis of the reaction mixture by gel electrophoresis is shown.
(C) Decreased thermal stability of o-phenanthroline-treated CAD. The Zn2+-removed CAD was prepared by dialysis against a buffer solution consisting of 20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 10 mM dithiothreitol, and 14 mM o-phenanthroline for 4 hr at 25°C. Zn2+-removed CAD and intact CAD were dialyzed against a buffer solution consisting of 10 mM sodium phosphate (pH 7.6), 100 mM NaCl, and 5 mM β-ME for 3 hr at 15°C. TM was analyzed with circular dichroism spectroscopy at 222 nm.
Molecular Cell  , DOI: ( /S (04) )

4. Figure 3 Aspect of Dimerization of CAD
(A) Intersubunit interactions mapped on sequence alignment. Sequences of all of the eight CAD orthologs available in public databases were aligned using the program ClustalW (Thompson et al., 1994), and four of them are shown. The purple circles below the sequences indicate the residues that are involved in the intersubunit interactions closer than 4.0 Å. The invariant residues in the eight CADs are shown in red letters. The black boxes indicate the Zn2+-coordinating residues. The secondary structure assignment is shown in two different colors to denote Domains C2 and C3. The numbers in parentheses indicate the omitted amino acids. The accession numbers for the eight CADs are M. musc (mouse, O54788), G. gall (chicken, AAL ), D. reri (zebra fish, AAF ), D. mela (fly, NP_ ), human (AAC ), rat (NP_ ), puffer fish (the Fugu genomic database), and mosquito (XP_ ).
(B) Loose dimerization interaction. The final 2Fo − Fc electron density map (2.6 Å, 1.0 σ) is shown for a part of the C2/C2 interface (left) and the C2/C3 interface (middle). The interactions between the α3 helices (left) and between the rest of the symmetry-related C2 domains (data not shown) are obviously loose. The interactions between Domains C2 and C3 are tight, except for the interactions mediated by Trp226 (Figure 2A). In contrast, the electron density throughout Domain C3 is well defined, as shown for a part of it (right), which includes an α helix.
Molecular Cell  , DOI: ( /S (04) )

5. Figure 4 Estimation of Molecular Weight
The 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 kDa, respectively.
Molecular Cell  , DOI: ( /S (04) )