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, 98 (25), 14250-5

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

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

M Renatus et al. Proc Natl Acad Sci U S A.

Abstract

A critical step in the induction of apoptosis is the activation of the apoptotic initiator caspase 9. We show that at its normal physiological concentration, caspase 9 is primarily an inactive monomer (zymogen), and that activity is associated with a dimeric species. At the high concentrations used for crystal formation, caspase 9 is dimeric, and the structure reveals two very different active-site conformations within each dimer. One site closely resembles the catalytically competent sites of other caspases, whereas in the second, expulsion of the "activation loop" disrupts the catalytic machinery. We propose that the inactive domain resembles monomeric caspase 9. Activation is induced by dimerization, with interactions at the dimer interface promoting reorientation of the activation loop. These observations support a model in which recruitment by Apaf-1 creates high local concentrations of caspase 9 to provide a pathway for dimer-induced activation.

Figures

Figure 1
Figure 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 = milli-absorbance units at 280 nm) eluted as a monomer (≈35 kDa for the ΔCARD forms and ≈48 kDa for the full-length form), but the majority of activity against the substrate Ac-LEHD-AFC (step curve; left ordinate, Rfu = relative fluorescence units) is present in the fraction corresponding to dimers (≈70 kDa for the ΔCARD forms and ≈100 kDa for the full length form). Insets demonstrate the integrity of the purified recombinant proteins run in SDS/PAGE 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 ΔCARD caspase 9 by glutaraldehyde in the absence (Upper) or presence (Lower) of 1.0 μM 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.
Figure 2
Figure 2
Schematic representation of ΔCARD caspase 9. (A) Caspase 9 is composed of two domains; the large subunit of each domain is colored gray and the small subunit is colored blue. The bound inhibitor molecule (gray), the side chains of the catalytic residue Cys-285 and the specificity determining residues Arg-341 and Trp-340 are in the highlighted ball and stick. Large deviations are seen in the environment of the catalytic and specificity-determining residues of the two domains (red). In the Left (active) domain, these residues adopt the conformations seen in other caspases, enabling binding of the inhibitor. In the Right (inactive) domain, the same residues are transposed from their catalytic conformation into a novel structure incapable of catalysis. The figure was made with MOLSCRIPT and rendered in RASTER3D, and secondary structure elements are assigned as for caspase 1 (17). (B) Stereoview of the activation loop. The region surrounding the activation loop in the active domain is superimposed on the equivalent region of the inactive domain. In the inactive domain (gray), displacement of the essential specificity determinant Arg341 disrupts the S1 and the S3 subsites; displacement of Trp-340 and Val-338 disrupts the S2 subsite; the backbone of Ser-339 and Arg-341 cannot form the short antiparallel β-strand with the substrate required for its proper alignment. Changes to the catalytic apparatus are equally substantial. Residues that provide hydrogen bonds to stabilize the oxyanion hole are perturbed, and the region at the terminus of the large subunit, which follows the catalytic Cys, are disordered and not defined by electron density. Transition to the active form (red) requires outward movement in the “priming bulge” to compensate the inward movement of Trp-340 and Arg-341. In the active domain residues after the catalytic Cys are well ordered up to Val-296. (C) Stereoview of the electron-density map defining the activation loop in the inactive domain. The loop is well defined by electron density, with an identical conformation in each copy of the inactive domain in the asymmetric unit. Hydrogen bonds are indicated by dotted lines.
Figure 3
Figure 3
The dimer interface. This view focuses on interactions that influence dimer formation. (A) Stereoview as in Fig. 2A showing the side chains of Phe-390 and Tyr-331 and the catalytic Cys-285 in the active (Left) and inactive (Right) catalytic domains, within the dimeric structure. (B) Stereoview of a superposition of the inactive monomer (gray) onto the active one (red). Rotation of Phe-390 about Cα–Cβ occurs in the transition from the inactive to the active conformation, allowing a compensatory rotation of Tyr-331 around its Cα–Cβ. This in turn may help to promote the catalytic conformation of Cys-285. The yellow side-chain represents the position of Phe-390′ in a hypothetical dimer made from two active catalytic domains. Note that it would clash with the active conformation of Phe-390, eliminating the possibility of having two active monomers in this dimer.
Figure 4
Figure 4
The mechanism of zymogen activation. Caspase 9 exists as a monomer at physiologic concentrations, with an exposed activation loop that renders the enzyme latent. (A) During dimerization, the activation loop (red) of the left domain is drawn into a pocket on the right domain. (B) The hydrophobic pocket, bordered by Pro-324, Phe-240f, and Phe-393 in the right domain, accepts Phe-334 and Phe-337 from the left domain. This locks into place the priming bulge (Ser-330–Ser-339) of the activation loop, enabling Trp-340 and Arg-341 to sink into their substrate-binding conformation, and simultaneously allowing hydrogen bonding to the segment following Cys-285. This transition generates catalytic potential in the left domain.

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