LZ-C9 and C9Holo have inverted activity for LEHD-AFC and pro-caspase-3 substrates. (A, C, E) Reaction progress curves of tetrapeptide substrate LEHD-AFC cleavage by LZ-C9, LZ-C9L or t-LZ-C9, respectively, and C9Holo at indicated concentrations. (B, D, F) Reaction progress curves of DEVD-AMC cleavage by caspase-3 activated by LZ-C9, LZ-C9L or t-LZ-C9, respectively, and C9Holo at the same concentrations as in A, C and E.

LZ-C9 and C9Holo have similar Km values for tetrapeptide substrate LEHD-AFC. (A, C, E, G) Reaction progress curves of LEHD-AFC cleavage at different concentrations by LZ-C9 (100 nM), C9Holo (100 nM full length caspase-9, 100 nM Apaf-1, 10 μM dATP and 500 nM cytochrome c), t-LZ-C9 (100 nM) and t-C9Holo (100 nM full length truncated caspase-9, 100 nM Apaf-1, 10 μM dATP and 500 nM cytochrome c), respectively. (B, D, F, H) Plots of initial velocities as a function of LEHD-AFC concentrations for LZ-C9, C9Holo, t-LZ-C9 and t-C9Holo, respectively. (I) Reaction progress curves of LEHD-AFC cleavage by caspase-9 at different Apaf-1 concentrations.

Potential molecular mechanisms of affinity enhancement of C9Holo for pro-caspase-3. The apoptosome, caspase-9 (C9) and pro-caspase-3 (C3) are shown in green, orange and purple, respectively. The inactive caspase-9 subunits are shown as rectangles. Asterisks denote contacts of pro-caspase-3 with either the apoptosome or caspase-9. For simplicity, only one pro-caspase-3 dimer is shown.

Engineering of LZ-C9, a dimeric caspase-9. (A) Schematic diagram of caspase-9, LZ-C9 and LZ-C9L. (B) A molecular model of LZ-C9 shown as a ribbon diagram. L151 residues of dimeric caspase-9 are shown as space filling models. Two orientations are show, left panel with the twofold axis horizontal and right panel with the twofold axis into the page. (C) Gel filtration profile of LZ-C9. Locations of molecular weight standards and C9ΔCARD are marked. SDS-PAGE of the peak fraction is shown. (D) Multiangle light scattering of LZ-C9. The trace in black corresponds to light scattering signals at 90°. The trace in dark red shows the variation in molecular mass determination as a function of peak position or elution volume.

Association of C9Holo with processed caspase-3 does not enhance caspase-3 activity. (A) Excess pro-caspase-3 was incubated with C9Holo and the mixture was subjected to gel-filtration. Aliquots of fractions were resolved by 15% SDS-PAGE followed by western blotting using antibodies against Apaf-1 (upper panel) and caspase-3 (lower panel). (B) Fractions were also assayed for their cleavage activity against DEVD-AMC. The initial velocities were plotted as a function of fraction numbers. Activities of fractions 10–13 were too high for initial velocities to be determined precisely and they were not shown. (C) Fraction 12 was serial diluted in 2-fold steps. Aliquots of each dilution were analyzed by 15% SDS-PAGE along with an aliquot of fraction 4 and blotted against caspase-3 (upper panel). They were also assayed for cleavage activity against DEVD-AMC. The initial velocities were plotted (lower panel). (D) Excess pro-caspase-3 was incubated with LZ-C9 under same conditions as in (A) and the mixture was subjected to gel-filtration. Fractions were resolved by 15% SDS-PAGE and blotted against caspase-3 (upper panel). Aliquots of fractions were assayed for cleavage activity against DEVD-AMC. The initial velocities were plotted as a function of fraction numbers (lower panel). Activities of fractions 11 and 12 were too high for initial velocities to be determined precisely and they were not shown.

C9Holo has much reduced Km for pro-caspase-3. (A, C, E) Reaction progress curves of pro-caspase-3 activation at different pro-caspase-3 concentrations by C9Holo (25 nM), LZ-C9 (200 nM) and t-C9Holo (25 nM), respectively. These were measured indirectly by the cleavage of the caspase-3 substrate DEVD-AMC. (B, D, F) Plots of initial velocities as a function of pro-caspase-3 concentrations for C9Holo, LZ-C9 and t-C9Holo, respectively.