Dimer Formation Drives the Activation of the Cell Death Protease Caspase 9

Dimer formation drives the activation of the cell death protease caspase 9 Martin Renatus, Henning R. Stennicke, Fiona L. Scott, Robert C. Liddington, and Guy S. Salvesen* Program in Apoptosis and Cell Death Research, The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037 Communicated by Erkki Ruoslahti, The Burnham Institute, La Jolla, CA, August 31, 2001 (received for review August 3, 2001) A critical step in the induction of apoptosis is the activation of the competent form, and here we describe studies that illustrate such apoptotic initiator caspase 9. We show that at its normal physio- a mechanism. logical concentration, caspase 9 is primarily an inactive monomer (zymogen), and that activity is associated with a dimeric species. At Materials and Methods the high concentrations used for crystal formation, caspase 9 is Recombinant Proteins. Full-length and ⌬CARD (caspase recruit- dimeric, and the structure reveals two very different active-site ment domain) human caspase 9 (lacking the first 138 residues) conformations within each dimer. One site closely resembles the were expressed in soluble form and purified from Escherichia catalytically competent sites of other caspases, whereas in the coli (5). A cleavage-defective mutant of caspase 9 containing second, expulsion of the ‘‘activation loop’’ disrupts the catalytic Asp͞Ala substitutions at the underlined residues PEPDA and machinery. We propose that the inactive domain resembles mo- DQLDA within the interdomain linker segment was generated, nomeric caspase 9. Activation is induced by dimerization, with expressed, and purified as described (5). Further mutations that interactions at the dimer interface promoting reorientation of the abrogated cleavage of caspase 9 were generated by replacing the activation loop. These observations support a model in which three acidic residues PEDES in the interdomain linker segment recruitment by Apaf-1 creates high local concentrations of caspase by Ala residues (21). The final noncleavable constructs con- 9 to provide a pathway for dimer-induced activation. tained all five Ala substitutions, and are designated as single- chain forms. apoptosis ͉ crystal structure ͉ zymogen activation Reagents. The polycaspase inhibitor benzoxycarbonyl-Val-Ala- Asp-fluoromethyl ketone (Z-VAD-FMK) and the caspase 9 he protease caspase 9 is the central participant in a multi- substrate acetyl-Leu-Glu-His-Asp-7-amino-4-trifluoromethyl component complex known as the apoptosome, which con- T coumarin (Ac-LEHD-AFC) were from Enzyme Systems Prod- trols cell deletion during development of the central nervous ucts, and the N-deblocked VAD-FMK was custom synthesized system and the apoptotic response to lethal cellular insults such by Enzyme Systems Products (Livermore, CA). The irreversible as ionizing radiation or chemotherapeutic drugs (1–3). The task inhibitor benzoxycarbonyl-Glu-Val-Asp-dichlorobenzylmethyl of caspase 9 is to generate the active forms of caspases 3 and 7 ketone (Z-EVD-Dcbmk) was a gift of Joe Wu, Idun Pharma- by limited proteolysis, and thereby transmit the apoptotic signal ceuticals (San Diego). to the execution phase. Most proteases exist in their biologically relevant locations in a latent form, and caspase 9 is no exception. Caspase 9 Assays. Hydrolysis of Ac-LEHD-AFC by caspase 9 was However, caspase 9 is unusual among its close relatives in that followed on a Molecular Devices fmax plate reader at 37°C. The proteolysis between the large and small subunit is not sufficient assay buffer was 10 mM Pipes͞0.1 M NaCl͞0.1 mM EDTA͞10 for conversion of the latent zymogen to the catalytic form (4, 5). mM DTT͞10% sucrose͞0.1% 3-[(3-cholamidopropyl)dimeth- In common with other initiators of proteolytic pathways (6–8), ylammonio]-1-propanesulfonate (CHAPS), pH 7.2. Cleavage of caspase 9 requires association with specific cofactors for its the substrate as a function of time was followed by monitoring activation, in this case an activated form of Apaf-1 within the the emission at 510 nm on excitation at 405 nm, and the initial apoptosome (1). velocity was determined from the linear portion of progress There are two fundamental ways in which protease zymogens curve. The enzymatically active concentration of caspase 9 was are maintained in an inactive conformation. In one case, exem- determined by titration with Z-VAD-FMK as described (22). plified by the lysosomal cysteine proteases (9) and the matrix metalloproteinases (10), the catalytic site is in an active confor- Gel Filtration. Gel filtration of caspase 9 derivatives was per- mation, but access is blocked; limited proteolysis removes the formed on a Amersham Pharmacia Superdex 200 column in 20 blocking N-terminal propeptide. In the second case, such as the mM Tris͞100 mM NaCl buffer (pH 8.0) with a flow rate of 1 chymotrypsin family exemplified by the coagulation serine pro- ml⅐minϪ1. The column dimensions were 30 ϫ 1 cm, and 0.5-ml teases, the catalytic site is not yet formed, and structural fractions were collected for analysis. rearrangements are required (11). These structural rearrangements can occur by limited proteolysis, or by cofactor binding Caspase-9 Crosslinking. Catalytically active ⌬CARD caspase 9, irrespective of proteolysis. Thus the executioner of the coagu- and ⌬CARD caspase 9 covalently inhibited with Z-VAD-FMK lation pathway, thrombin, is activated simply by proteolysis (12), were dialyzed against 100 mM phosphate buffer (pH 7.5), and but the initiator of the extrinsic branch of the coagulation the protein concentration was adjusted to 2.4 ␮M. The crosslink- pathway, factor VII, is inactive until bound by its cofactor (tissue factor; ref. 13). The structure of active caspases 1, 3, 7, and 8 with peptidyl Abbreviations: Z, benzoxycarbonyl; AFC, 7-amino-4-trifluoromethyl coumarin; FMK, fluoromethyl ketone; Dcbmk, dichlorobenzyl methylketone. inhibitors occupying their catalytic sites has clarified the com- Data deposition: The caspase 9 coordinates have been deposited in the Protein Data Bank, mon mechanism of the enzymes (14–20), yet little was known of www.rcsb.org (PDB ID code 1JXQ). the molecular basis of activation of the latent forms, nor the *To whom reprint requests should be addressed. E-mail: [email protected]. molecular mechanism of maintenance of the inactive zymogens. The publication costs of this article were defrayed in part by page charge payment. This Because proteolysis is not required for activation of caspase 9, article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. there must exist another mechanism to achieve the catalytically §1734 solely to indicate this fact. 14250–14255 ͉ PNAS ͉ December 4, 2001 ͉ vol. 98 ͉ no. 25 www.pnas.org͞cgi͞doi͞10.1073͞pnas.231465798 Fig. 1. Identification of the active form of caspase 9. (A) Gel filtration of caspase 9 on a Amersham Pharmacia Superdex 200 column reveals that most of the protein for each form (smooth curge; right ordinate, mAU ϭ milli-absorbance units at 280 nm) eluted as a monomer (Ϸ35 kDa for the ⌬CARD forms and Ϸ48 kDa for the full-length form), but the majority of activity against the substrate Ac-LEHD-AFC (step curve; left ordinate, Rfu ϭ relative fluorescence units) is present in the fraction corresponding to dimers (Ϸ70 kDa for the ⌬CARD forms and Ϸ100 kDa for the full length form). Insets demonstrate the integrity of the purified recombinant proteins run in SDS͞PAGE before gel filtration. The arrow marks the position of the protein peak for two chain caspase 9 inhibited by Z-VAD-FMK. (B) Crosslinking of ⌬CARD caspase 9 by glutaraldehyde in the absence (Upper) or presence (Lower)of1.0␮M Z-VAD-FMK. The gray triangle above the gels indicates the glutaraldehyde concentration (ranging from 500 to 0.032 mM). The majority of caspase 9 material is a monomer, and inhibitor binding forces the dimeric form. Thus, it appears that the inhibitor either drives or traps the dimeric form of caspase 9, indicating that the dimer is the active form of the enzyme. ing reaction was performed by incubating 50 ␮l of the protein ing kits from Hampton Research (Riverside, CA), and crystals solution with 50 ␮l of aqueous glutaraldehyde solutions at where obtained from 0.1 M Mes (pH 6.5), 12% PEG 20,000. BIOCHEMISTRY different concentrations (500 mM to 32 ␮M) for 5 min at 23°C. Two-chain ⌬CARD caspase 9 inhibited with Z-EVD-Dcbmk The reaction mixture was reduced for 30 min at 37°C with 20 ␮l crystallized under the same conditions and diffracted to 2.8 Å. of 2 M NaBH4. The crosslinked protein was precipitated by The crystal symmetry and the unit cell dimensions where adding 50 ␮l of 80% trichloroacetic acid, washed, and run in identical to those of the first crystal. Molecular replacement was SDS͞PAGE in gels of 8–18% linear acrylamide gradient with a performed with AMoRe (25) and caspase 3 as search model, 2-amino-2-methyl-1,3-propanediol͞glycine͞HCl system (23). map interpretation was done in MAIN (26), data processing and Samples for SDS͞PAGE were boiled in SDS sample buffer scaling were done in DENZO͞SCALEPACK (27), and crystallo- containing 50 mM DTT for 5 min and then loaded onto the graphic refinement (simulated annealing, positional- and stacking gel. temperature-factor refinement) in Crystallography & NMR System (28). Noncrystallographic symmetry restraints were used N-Terminal Sequencing of Protein Samples. For N-terminal se- between the two dimers in the asymmetric unit, but not between quencing, protein samples were resolved by SDS͞PAGE or the catalytic in each dimer. In the Ramachandran plot of the nondenaturing PAGE and transferred to Immobilon-P mem- 2.8-Å structure final model 90.2% of the ␺͞␾ angles fall in the brane (Millipore) by electroblotting (24). The membrane was core region and 9.8% in the additional allowed region.

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