Modifications to a common phosphorylation network provide individualized control in caspases

Abstract

Caspase-3 activation and function have been well-defined during programmed cell death, but caspase activity, at low levels, is also required for developmental processes such as lymphoid proliferation and erythroid differentiation. Post-translational modification of caspase-3 is one method used by cells to fine-tune activity below the threshold required for apoptosis, but the allosteric mechanism that reduces activity is unknown. Phosphorylation of caspase-3 at a conserved allosteric site by p38-MAPK (mitogen-activated protein kinase) promotes survival in human neutrophils, and the modification of the loop is thought to be a key regulator in many developmental processes. We utilized phylogenetic, structural, and biophysical studies to define the interaction networks that facilitate the allosteric mechanism in caspase-3. We show that, within the modified loop, Ser¹⁵⁰ evolved with the apoptotic caspases, whereas Thr¹⁵² is a more recent evolutionary event in mammalian caspase-3. Substitutions at Ser¹⁵⁰ result in a pH-dependent decrease in dimer stability, and localized changes in the modified loop propagate to the active site of the same protomer through a connecting surface helix. Likewise, a cluster of hydrophobic amino acids connects the conserved loop to the active site of the second protomer. The presence of Thr¹⁵² in the conserved loop introduces a "kill switch" in mammalian caspase-3, whereas the more ancient Ser¹⁵⁰ reduces without abolishing enzyme activity. These data reveal how evolutionary changes in a conserved allosteric site result in a common pathway for lowering activity during development or a more recent cluster-specific switch to abolish activity.

Keywords: X-ray crystallography; allosteric regulation; apoptosis; biophysics; caspase; computational biology; fluorescence; molecular dynamics; protein evolution.

Figure 1.

A, model of caspase-3 activation, regulation by XIAP binding, phosphorylation, and degradation. The caspase protomer is represented by one LS and one SS. In the zymogen, the subunits in the protomer are covalently connected by the IL. In the mature caspase, the IL is cleaved to yield the mature caspase protomer. Following cleavage, the IL provides two of the five active site loops, called L2 and L2′ (see B and Fig. 2). Both the zymogen and the mature caspase-3 are considered a dimer of protomers, but the IL is cleaved in the mature enzyme. Phosphorylation of the zymogen inhibits maturation, whereas phosphorylation of the mature caspase inhibits enzyme activity. Binding of XIAP inhibits the mature caspase and leads to proteasomal degradation. B, secondary structural elements mapped onto the caspase-3 sequence. This figure was generated using Polyview-2D (67) and the structure of human caspase-3 (PDB entry 2J30). β-Strands 1–6 (green), α-helices 1–5 (red; H1–H5), and active site loops (blue) L1–L4 and L2′ are indicated (see also Fig. 2). The arrow indicates the site of cleavage (Asp¹⁷⁵) of the IL to yield loops L2 and L2′ of the mature protomer.

Figure 2.

A, structure of caspase-3 (PDB entry 2J30). The LS and SS (see Fig. 1) fold into a single domain with a central six-stranded β-sheet core (β1–β4 contributed by the LS and β5 and β6 contributed by the SS; labeled in protomer 1) and five external α-helices (H1–H3 contributed by the LS and H4 and H5 contributed by the SS). Five loops comprise the active site of each protomer, where L1, L2, L3, and L4 are contributed by one protomer and L2′ is contributed by the second protomer of the dimer (see Fig. 1). The loop bundle refers to interactions between L4, L2, and L2′, which stabilize the active site. B, interactions in the H3CL (helix-3 C-terminal loop, green) with helix-2 (H2, blue/brown) and the loop bundle (L2, red; L2′, cyan). Hydrogen bonds from Ser¹⁵⁰ and Thr¹⁵² are shown as dashed lines. C, active-site loop L4 (brown) is stabilized, in part, by hydrogen bonds from Ser²⁴⁹, which contributes to substrate binding through interactions with Phe²⁵⁰ and the aspartate in the P4 position of the substrate. Inhibitor refers to DEVD-chloromethylketone used during crystallization of the wildtype enzyme. Note that, for clarity, the active site in C is rotated 180° relative to the orientation in A.

Figure 3.

A and B, collapsed phylogenetic trees of 175 caspase-3 sequences, bootstrapped 100 times, with chondrichthyes (cartilaginous fish) as the out group. Groups are labeled according to their phylogenetic class or subclass. A, the most probable ancestor of position Ser¹⁵⁰ at the nodes shows that Ser¹⁵⁰ is highly conserved from chondrichthyes to humans. B, the most probable ancestor of position Thr¹⁵². Metatherian mammals are not collapsed to show that threonine at position 152 evolved early in the mammalian lineage, between Ornithorhynchus anatinus (platypus) and Monodelphis domestica (opossum). A and B are expanded in Fig. S3B. C, multiple-sequence alignment for each caspase using every known caspase sequence from NCBI using WebLogo (68). Numbers below each position refer to caspase-3 numbering. D, salt bridge between Glu¹⁷⁶ and Arg²⁷¹ in caspase-7 that spans across the dimer interface.

Figure 4.

A, whole protein digest. Lane 1, protein molecular weight standards; lane 2, 10 μm WT caspase-3 control; lane 3, 75 μm caspase-7 (C186S) control; lane 4, WT+CP7 (pH 7.5); lane 5, WT+CP7 (pH 6); lane 6, S150A+CP7 (pH 7.5); lane 7, S150A+CP7 (pH 6); lane 8, S150D+CP7 (pH 7.5); lane 9, S150D+CP7 (pH 6); lane 10, T152V+CP7 (pH 7.5); lane 11, T152V+CP7 (pH 6); lane 12, T152D+CP7 (pH 7.5); lane 13, T152D+CP7 (pH 6); lane 14, protein molecular weight standards. In A, CP7 refers to caspase-7. B, model of pH-dependent oligomeric states of caspase-3 from dimer to unfolded protomer. C and D, AEW following excitation at 295 nm in the native state (C) and unfolded in 8 m urea (D). E, pH gradient versus relative activity for WT and caspase-3 (Ser¹⁵⁰ or Thr¹⁵²). Error bars, S.D. from three experiments. F, AEW following excitation at 295 nm versus pH for WT, Ser¹⁵⁰, and Thr¹⁵² caspase-3 proteins. The following symbols were used: WT (♦), S150A (○), S150D (□), T152V (□), and T152D (●).

Figure 5.

A–G, hydrogen bonds and electrostatic interactions in the H3CL of WT (gray) superimposed to each caspase-3 mutant (green). Blue spheres, conserved waters as described under “Experimental procedures” (24). A, S150A; B, S150D; C, S150E; D, S150Y; E, T152V; F, T152A; G, T152D. For each panel, black dashes represent hydrogen bonds in WT caspase-3, and red dashes represent hydrogen bonds in the mutant.

Figure 6.

A, representative frame of a molecular dynamics simulation of S150E (green) compared with the X-ray crystal structure of wildtype caspase-3 (PDB code 2J30) showing movement of surface strands β¹ and β² toward the catalytic residue His¹²¹. Black dashes, initial distance between the side chain of Glu¹²³ and the catalytic histidine (∼11 Å); red dashes, distance between Glu¹²³ and His¹²¹ in the mutant (∼3.5 Å). B, positions of the catalytic dyad (His¹²¹ and Cys¹⁶³) and of Glu¹²³ shown as 200 frames from the 50-ns MD simulation.

Figure 7.

A, location of histidine residues in caspase-3. Active-site loops L1, L4, and L2′ each contain one histidine (His⁵⁶, His²⁵⁷, and His¹⁸⁵, respectively). Two histidine residues at the C terminus (His²⁷⁷ and His²⁷⁸) are not labeled. B, near the H3CL, Ser¹⁵⁰ hydrogen-bonds to His¹⁰⁸. C, in wildtype caspase-3, the dimer is stabilized by electrostatic interactions between helix-5 and helix-5′ across the dimer interface, facilitated by Glu²³¹, His²³⁴, and Glu²⁷² of each protomer. The prime symbol indicates amino acids of the second protomer.