Nucleotide status and hydrolytic properties of Apaf-1. A, Coomassie-stained SDS-PAGE showing the purity of the proteins used in the assays. Lane M, molecular weight marker; lane 1, horse cytochrome c; lane 2, caspase-9; lane 3, Apaf-1. B, Apaf-1 purified from Sf21 insect cells contains ADP. Absorption curves corresponding to reference nucleotides are shown as black dotted lines; the absorption curve of the nucleotide isolated from purified Apaf-1 is shown as green solid line. C, Apaf-1 hydrolyzes ATP and dATP at a low rate. 10 μm Apaf-1 was incubated in buffer A at 30 °C with 1 mm ATP and dATP, respectively. Samples taken at different time points were analyzed on an HPLC system to detect the formed nucleoside diphosphate. By comparison with reference samples, the respective peak area (supplemental Fig. S1) was converted into concentration (green or yellow dots). As controls, the same was done with samples containing nucleotide but no Apaf-1 (green or yellow open squares) or Apaf-1 together with the nonhydrolyzable ATP analog AppNHp (blue dots). Mean averages of triplicate measurements are plotted against time. The resulting linear fits represent an ATPase activity of 4.33 ± 0.13 ATP molecules per molecule of Apaf-1 per h and a dATPase activity of 4.83 ± 0.04 dATP molecules per molecule of Apaf-1 per h. D, Apaf-1 is able to exchange ADP for dATP in the absence of cytochrome c. 500 μg of Apaf-1 were incubated with 1 mm dATP at 20 °C for 3 h. After separation of the protein from excess nucleotide by gel filtration, the peak fractions were concentrated and analyzed by HPLC. Absorption curves corresponding to reference nucleotides are shown as black dotted lines, and the absorption curve of the nucleotide isolated from Apaf-1 is shown as green solid line. mAU, milliabsorbance units.

Apoptosome formation using recycled Apaf-1. Recycled ADP-bound Apaf-1 remains in the monomeric state if no cofactors are supplied (black curve) or if only cytochrome c is added (yellow curve). As naive Apaf-1, recycled ADP-bound Apaf-1 requires both exogenous ATP and cytochrome c for apoptosome formation (blue curve). On the other hand, recycled AppNHp-bound Apaf-1 only requires cytochrome c for apoptosome formation (pink curve). mAU, milliabsorbance units.

Cofactor requirements for apoptosome formation and caspase-9 activation. A, nucleotide dependence of apoptosome formation. Apaf-1 at a concentration of 10 μm was incubated at 4 °C overnight with a 5-fold excess of cytochrome c and 1 mm nucleotide. The reaction mixtures were analyzed by analytic gel filtration. Apoptosome formation is indicated by strong decrease of the elution volume. Addition of a nucleoside triphosphate (blue, ATP; yellow, dATP; pink, AppNHp; green, GTP) leads to formation of apoptosomes. Incubation with ADP (black curve) or dADP (gray curve) has no effect. The elution volumes of globular marker proteins are indicated. B, nucleotide dependence of caspase-9 activation. Apaf-1 at a concentration of 0.4 μm was incubated at 30 °C for 10 min with 2 μm cytochrome c, 0.2 μm caspase-9, and nucleotide as indicated. Ac-LEHD-AFC was added to a final concentration of 200 μm, and the reaction was fluorometrically monitored. Measurements were done in triplicate; error bars are standard deviations. Control 1 contained no nucleotide; control 2 contained only caspase-9. C, stimulation of caspase-9 activity by the apoptosome is independent of the nucleotide used for assembly. Apoptosomes were assembled using ATP, dATP, AppNHp, and GTP, respectively, and isolated via gel filtration. Isolated apoptosomes equivalent to 0.4 μm Apaf-1 monomer were incubated with 0.2 μm caspase-9 at 30 °C for 10 min. Ac-LEHD-AFC was added to a final concentration of 200 μm, and the reaction was fluorometrically monitored. Measurements were repeated five times; error bars are standard deviations. D, only at very low nucleotide concentrations is dATP more efficient for caspase-9 activation than ATP. Assay conditions are as in B. mAU, milliabsorbance units.

Model of the mechanism of apoptosome formation. A, autoinhibited Apaf-1. Two blocking fixtures secure the closed state as follows: interactions between the WD40 propellers and the NOD (indicated by a gray rectangle protruding from one of the gray ovals) and interactions between subdomains of the NOD (indicated by tight arrangement of the blue oval and the blue triangle). B, Apaf-1 liganded with ATP. The presence of the γ-phosphate weakens subdomain interactions within the NOD (indicated by a gap between the blue oval and the blue triangle). Cyt. c, cytochrome c. C, Apaf-1 liganded with cytochrome c. Blocking interactions between WD40 propellers and the NOD are released. D, Apaf-1 liganded with cytochrome c and ATP. Both blocking fixtures are removed. E, open Apaf-1 ready to assemble into the apoptosomal heptamer.