Mechanistic insights into CED-4-mediated activation of CED-3

Abstract

Programmed cell death in Caenorhabditis elegans requires activation of the caspase CED-3, which strictly depends on CED-4. CED-4 forms an octameric apoptosome, which binds the CED-3 zymogen and facilitates its autocatalytic maturation. Despite recent advances, major questions remain unanswered. Importantly, how CED-4 recognizes CED-3 and how such binding facilitates CED-3 activation remain completely unknown. Here we demonstrate that the L2' loop of CED-3 directly binds CED-4 and plays a major role in the formation of an active CED-4-CED-3 holoenzyme. The crystal structure of the CED-4 apoptosome bound to the L2' loop fragment of CED-3, determined at 3.2 Å resolution, reveals specific interactions between a stretch of five hydrophobic amino acids from CED-3 and a shallow surface pocket within the hutch of the funnel-shaped CED-4 apoptosome. Structure-guided biochemical analysis confirms the functional importance of the observed CED-4-CED-3 interface. Structural analysis together with published evidence strongly suggest a working model in which two molecules of CED-3 zymogen, through specific recognition, are forced into the hutch of the CED-4 apoptosome, consequently undergoing dimerization and autocatalytic maturation. The mechanism of CED-3 activation represents a major revision of the prevailing model for initiator caspase activation.

Keywords: CED-3; CED-4; apoptosis; caspase activation; programmed cell death (PCD).

Figure 1.

CED-4-mediated CED-3 activation is a central step to initiate PCD in C. elegans. (A) Four genes—egl-1, ced-9, ced-4, and ced-3—act in a linear fashion to control the onset of PCD in C. elegans. (B) Current model of the linear PCD pathway. In normal cells, CED-4 is sequestered by CED-9, unable to activate the cell-killing caspase CED-3. Upon cell death stimuli, EGL-1 is activated and binds to CED-9. Binding by EGL-1 causes marked conformational changes in CED-9, resulting in the release of CED-4. The released CED-4 dimer oligomerizes to form the CED-4 apoptosome, which in turn facilitates the activation of CED-3. How CED-4 binds CED-3 remains unclear, hindering a mechanistic understanding of CED-3 activation. (C) Structure of the two-chain CED-3 (residues 198–374, 389–503, and C358S). Despite being a monomer in solution, CED-3 was crystallized as an asymmetric dimer, likely due to the high concentrations required for crystallization.

Figure 2.

The L2′ loop of CED-3 is required for stable association between CED-3 and the CED-4 apoptosome. (A) The L2′ loop is required for CED-3 to associate with the CED-4 apoptosome. Gel filtration analysis reveals that removal of the L2′ loop, but not the L2 loop, in the two-chain CED-3 (residues 198–374, 389–503, and C358A) resulted in loss of binding to the CED-4 apoptosome. (B) Deletion of the L2′ loop in CED-3 results in loss of CED-4-mediated stimulation of CED protease activity. (C) Missense mutations in the L2′ loop of CED-3 leads to abrogation of CED-4-mediated stimulation of CED protease activity. The three missense mutations L391D, F392D, and N393D target three consecutive amino acids in the L2′ loop of CED-3.

Figure 3.

The L2′ loop of CED-3 is sufficient for stable association with the CED-4 apoptosome. (A) Replacement of the L2′ loop (residues 316–331) in caspase-9 by that in CED-3 (residues 389–404) allowed weak interaction between the caspase-9 variant and the CED-4 apoptosome. Contiguous fractions from gel filtration were visualized on Coomassie-stained SDS-PAGE gels. Red arrows denote a small fraction of the caspase-9 variant that had been shifted to earlier fractions. The wild-type (WT) caspase-9 controls are shown in the top two panels. (B) Replacement of the L2′ loop (residues 316–331) in caspase-9 by that in CED-3 (residues 389–404) resulted in higher protease activity in the presence of CED-4. LEHD-AFC was used as the substrate.

Figure 4.

Crystal structure of the CED-4 apoptosome bound to a L2′ loop fragment of CED-3. (A) Overall structure of the CED-4 apoptosome bound to a L2′ loop fragment of CED-3, viewed from the wide opening side of the funnel-shaped CED-4 apoptosome. The CED-4 apoptosome binds eight molecules of the L2′ loop fragment. (B) A close-up view of the CED-3 L2′ loop fragment bound to CED-4. Seven contiguous amino acids (residues 390–396) were unambiguously assigned. (C) Confirmation of the L2′ sequence assignment by selenium anomalous signals. The two Leu residues in the L2′ loop fragment, Leu391 and Leu395, were singly or doubly mutated to Met for generation of selenium anomalous signals (colored magenta and shown at 3σ). 2Fo − Fc electron density (light blue) for the L2′ loop fragment is displayed at 1.0 sec. (D) A cut-through section of the side view of the CED-4 apoptosome bound to the CED-3 L2′ fragment. (E) The neighboring L2′ loop fragments within the CED-4 apoptosome are separated by distances ranging from 30 to 83 Å. This and all other structural figures were prepared using PyMol (http://www.pymol.org).

Figure 5.

Recognition of the CED-3 L2′ loop by CED-4 is required for the stable association of the CED-3–CED-4 complex. (A) A close-up view on the predominantly hydrophobic interface between CED-4 and the CED-3 L2′ loop fragment. The interface involves four hydrophobic residues from CED-4 (Val382, Leu393, Phe463, and Leu464) and two from CED-3 (Phe392 and Leu391). (B) The mutation A394W in CED-4 led to weakened interaction between CED-3 and the CED-4 apoptosome, as judged by gel filtration. Compared with the wild-type (WT) CED-4 (left panels), the CED-4 A394W variant exhibited a markedly diminished ability to interact with CED-3 (residues 198–503 and C358S) (right panels).

Figure 6.

Recognition of the CED-3 L2′ loop by CED-4 is required for both efficient CED-3 activation and enhanced protease activity. (A) Autocatalytic processing of the CED-3 zymogen is greatly facilitated by the wild-type (WT) CED-4 but not the CED-4 A394W variant. Shown here is a representative autoradiograph. The full-length wild-type CED-3 protein was in vitro translated in the presence of ³⁵S-Met. (B) The mutation A394W in the CED-4 hydrophobic pocket nearly abrogated the ability of the CED-4 apoptosome to stimulate the protease activity of the processed CED-3. DEVD-AMC was used as the substrate.

Figure 7.

A mechanistic model of the regulation of PCD initiation in C. elegans. In nonapoptotic cells, CED-4 is sequestered by CED-9, unable to form an oligomeric apoptosome for CED-3 activation. In dying cells, EGL-1 is transcriptionally activated, binds CED-9, and causes allosteric changes in CED-9 that no longer allow CED-4 association. The freed CED-4 forms an apoptosome, which recruits the CED-3 zymogen. The caspase domains of two CED-3 molecules are likely bound within the hutch of the funnel-shaped CED-4 apoptosome, where dimerization and subsequent autoprocessing of CED-3 are greatly facilitated. The processed CED-3 remains bound to the CED-4 apoptosome as a holoenzyme, mainly via interaction between the L2′ loop of CED-3 and a hydrophobic surface pocket within the hutch of the CED-4 apoptosome.