1. Mechanisms of Caspase Activation and Inhibition during Apoptosis
Yigong Shi 
Molecular Cell 
Volume 9, Issue 3, Pages (March 2002)
DOI: /S (02)
Copyright © 2002 Cell Press Terms and Conditions

2. Figure 1 Schematic Diagram of the Mammalian Caspases
Except caspase-11 (mouse), -12 (mouse), and -13 (bovine), all listed caspases are of human origin. Their phylogenetic relationship (left) appears to correlate with their function in apoptosis or inflammation. The initiator and effector caspases are labeled in purple and red, respectively. The position of the first activation cleavage (between the large and small subunits) is highlighted with a large arrow while additional sites of cleavage are represented by medium and small arrows. In contrast to other protease zymogens, removal of the prodomain of a caspase is unnecessary for its catalytic activity. The four surface loops (L1-L4) that shape the catalytic groove are indicated. The catalytic residue Cys is shown as a red line at the beginning of loop L2. This diagram is scaled according to the lengths of caspases and the location of functional segments.
Molecular Cell 2002 9, DOI: ( /S (02) )
Copyright © 2002 Cell Press Terms and Conditions

3. Figure 2 Structural Features of Caspases
(A) Representative structure of an inhibitor-bound caspase-3 (PDB code 1DD1). The small and large subunits are colored orange and blue, respectively. The bound peptide inhibitor is shown in pink. The four surface loops that constitute the catalytic groove of one heterodimer are labeled. The apostrophe denotes the other heterodimer. Note that L2′ stabilizes the active site of the adjacent heterodimer.
(B) The active site conformation of all known caspases is conserved. Of the four loops, L1 and L3 are relatively constant while L2 and L4 exhibit greater variability. The catalytic residue Cys is highlighted in red.
(C) Schematic diagram of the substrate-binding groove. L1 and L4 constitute two parallel sides of the groove while L3 serves as the base. L2, harboring the catalytic residue Cys, is positioned at one end of the groove, poised for catalysis. L2′ plays a critical role by stabilizing the conformation of the L2 and L4 loops.
(D) Conformation of the L4 loop in five known caspase structures. The L4 loop dictates the P4 specificity of the substrate. Short L4 loops (caspase-1, -8, and -9) allow bulkier and hydrophobic residue in the P4 position whereas extended L4 loops (caspase-3 and -7) prefer Asp. Coloring scheme is the same as (B). Figures were prepared using MOLSCRIPT (Klaulis, 1991) and GRASP (Nicholls et al., 1991).
Molecular Cell 2002 9, DOI: ( /S (02) )
Copyright © 2002 Cell Press Terms and Conditions

4. Figure 3 Mechanisms of Procaspase-7 Activation and Substrate Binding
(A) Structure of a procaspase-7 zymogen (PDB code 1K86). Compared to that of the inhibitor-bound caspase-7, the conformation of the active site loops does not support substrate binding or catalysis. The L2′ loop, locked in a closed conformation by covalent linkage, is occluded from adopting its productive and open conformation.
(B) Structure of an active and free caspase-7 (PDB code 1K88). The active site loops are still flexible. Despite an interdomain cleavage, the L2′ loop still exists in the closed conformation, indicating an induced-fit mechanism for binding to inhibitors/substrates.
(C) Comparison of the conformation of the active site loops. Compared to the procaspase-7 zymogen or the free caspase-7, the L2′ loop is flipped 180o in the inhibitor-bound caspase-7 to stabilize loops L2 and L4.
Molecular Cell 2002 9, DOI: ( /S (02) )
Copyright © 2002 Cell Press Terms and Conditions

5. Figure 4
Mechanisms of IAP-Mediated Inhibition of Effector Caspases and p35-Mediated Pan-Caspase Inhibition
(A) Superposition of the structures of caspase-3 (brown) and -7 (green) together with their bound XIAP fragments colored blue and pink, respectively. The interactions primarily occur between a linker segment N-terminal to the BIR2 domain of XIAP and the active site of caspase-3 or -7 (boxed region). The small red circle on BIR2 marks where the Smac N-terminal tetrapeptide binds and the large red circle indicates a likely second interface with Smac.
(B) Close-up view of the active sites of caspase-3 and -7 bound to their respective XIAP fragments. Two hydrophobic residues of XIAP, Leu141 and Val146, make multiple van der Waals interactions with a conserved hydrophobic pocket on caspase-3 or -7. Asp148 of XIAP, occupying the S4 pocket, hydrogen bonds to neighboring residues in caspase-3 or -7.
(C) Close-up view of the covalent inhibition of caspase-8 by p35. The thioester intermediate is shown between Asp87 of p35 and Cys360 (active site residue). The N terminus of p35 restricts solvent access to this intermediate.
Molecular Cell 2002 9, DOI: ( /S (02) )
Copyright © 2002 Cell Press Terms and Conditions

6. Figure 5 A Conserved IAP-Binding Tetrapeptide Motif
(A) Structure of the mature Smac. The disordered N-terminal residues are shown as dotted lines.
(B) Close-up view of the Smac N-terminal tetrapeptide binding to the BIR3 surface groove. The BIR3 domain is shown either by electrostatic potential (left panel) or in ribbon diagram (right panel) to highlight the interactions. The amino and carbonyl groups of the N-terminal Ala make several hydrogen bonds to conserved residues in XIAP.
(C) A conserved motif of IAP-binding tetrapeptides. The tetrapeptide motif has the consensus sequence A-(V/T/I)-(P/A)-(F/Y/I/V/S). The Drosophila proteins have an additional binding component (conserved 6th–8th residues).
(D) A conserved IAP-binding mode from mammals to fruit flies. The structure of DIAP1-BIR2 is superimposed with that of the XIAP-BIR3 domain, with their corresponding bound peptides Hid (pink), Grim (blue), and Smac/DIABLO (green).
Molecular Cell 2002 9, DOI: ( /S (02) )
Copyright © 2002 Cell Press Terms and Conditions

7. Figure 6
Schematic Diagram of the Mechanisms of Caspase Activation and Inhibition
(Left box) The tetrapeptide motif (red arrows), present in the N termini of Smac and the small subunit of caspase-9, binds to a surface groove on the BIR domains of IAPs (XIAP, c-IAP1, c-IAP2, and Livin/ML-IAP). This motif is responsible for both caspase-9 inhibition through binding BIR3 and the relief of inhibition through displacement of the caspase-9 tetrapeptide with a Smac tetrapeptide. Positive feedback cleavage by caspase-3 or -7 permanently removes IAP-mediated inhibition of caspase-9. The released caspase-9 linker peptide can antagonize IAP-mediated inhibition of other caspases.
(Right box) On the other hand, a linker segment N-terminal to the BIR2 domain of XIAP is primarily responsible for caspase-3 inhibition. Smac binding to the BIR2 domain may make this linker segment unavailable for binding to caspase-3. For clarity, only BIR domains but not the full-length IAPs are shown in this figure.
Molecular Cell 2002 9, DOI: ( /S (02) )
Copyright © 2002 Cell Press Terms and Conditions

8. Figure 7 Structure of Caspase-9 and the Apoptosome
(A) Two views of the heptameric apoptosome comprising Apaf-1, Cyt. c, and dATP. The CARD domains are located in the central hub while the WD40 repeats were mapped to the end of the spoke.
(B) Structure of the inhibitor-bound caspase-9. Only half of the caspase-9 dimer contains a well-formed active site (shown in green), covalently bound by an inhibitor. The active site loops in the other half, shown in pink, do not form a functional catalytic groove.
(C) A proposed model for the activation of caspase-9. The apoptosome-bound caspase-9 exists in an inactive conformation. The high local concentrations of caspase-9 within the apoptosome drive the recruitment of additional caspase-9 monomers, which are activated upon dimerization.
Molecular Cell 2002 9, DOI: ( /S (02) )
Copyright © 2002 Cell Press Terms and Conditions