Zinc-mediated allosteric inhibition of caspase-6

Abstract

Zinc and caspase-6 have independently been implicated in several neurodegenerative disorders. Depletion of zinc intracellularly leads to apoptosis by an unknown mechanism. Zinc inhibits cysteine proteases, including the apoptotic caspases, leading to the hypothesis that zinc-mediated inhibition of caspase-6 might contribute to its regulation in a neurodegenerative context. Using inductively coupled plasma optical emission spectroscopy, we observed that caspase-6 binds one zinc per monomer, under the same conditions where the zinc leads to complete loss of enzymatic activity. To understand the molecular details of zinc binding and inhibition, we performed an anomalous diffraction experiment above the zinc edge. The anomalous difference maps showed strong 5σ peaks, indicating the presence of one zinc/monomer bound at an exosite distal from the active site. Zinc was not observed bound to the active site. The zinc in the exosite was liganded by Lys-36, Glu-244, and His-287 with a water molecule serving as the fourth ligand, forming a distorted tetrahedral ligation sphere. This exosite appears to be unique to caspase-6, as the residues involved in zinc binding were not conserved across the caspase family. Our data suggest that binding of zinc at the exosite is the primary route of inhibition, potentially locking caspase-6 into the inactive helical conformation.

FIGURE 1.

Zinc is a potent inhibitor of caspase-6 activity. A, expression constructs showing sites of cleavage (gap) in full-length (FL) wild-type (WT), full-length zymogen (FL C163S), and constitutive two-chain (CT) caspase-6. Protein domains include the prodomain (gray circle), large subunit (gray bar), intersubunit linker (ISL, black line), and small subunit (black bar) with the active site cysteine (black C) mutated to serine (black S) in the large subunit. Residue numbers for each domain are indicated. RBS indicates ribosome-binding sites for initiation of translation in the CT construct. B, caspase-6-mediated cleavage of caspase-6 FL C163S zymogen. Metal inhibition was tested in an electrophoretic mobility gel-based assay as fragments produced from active caspase-6 (ΔND179CT) mediated cleavage of the full-length C163S zymogen that serves as the substrate. Fragments from cleavage include the following: full-length (FL), FL lacking the N-terminal prodomain (ΔN-FL), large (Lg), and small subunits (Sm) with the amino acids present in those bands (subscripts) labeled. Zinc is the only metal cation that inhibits caspase-6 activity. C, Caspase-6-mediated cleavage of caspase-6 FL C163S zymogen was tested at various zinc concentrations in an electrophoretic mobility gel-based assay. The readout for the assay was caspase-6 fragments produced from active caspase-6-mediated cleavage at the intersubunit linker of the zymogen. Proteolytic fragments are labeled as in B. D, in-gel IC₅₀ band quantification measuring band intensities. The full-length procaspase-6 FL C163S was set as 100% (uncleaved). Band quantification was measured as disappearance of these bands and appearance of cleavage bands in the gel. The IC₅₀ value is reported as the zinc concentration at which 50% of FL C163S has disappeared and 50% of the large subunit has appeared. The IC₅₀ value for zinc inhibition is 0.37 μm. E, dose-response of caspase-6 inhibition in the presence of zinc and a fluorogenic substrate (Ac-VEID-AMC). IC₅₀ was fit from independent duplicate metal titration on three separate days.

FIGURE 2.

Caspase-6 crystals bind zinc. An x-ray fluorescence scan measuring intensity (e) as a function of x-ray energy (eV) shows absorption at the zinc energy edge, 9673.92 eV, indicating that zinc is bound in caspase-6 crystals even after 10 min of washing.

FIGURE 3.

Zinc-bound caspase-6 retains fold. A, zinc-bound caspase-6 retains the same fold as zinc-free caspase-6, with one zinc bound per monomer. The zinc (blue sphere), zinc ligands Lys-36, Glu-244, and His-287 (sticks), and a water (red sphere) are shown. B, superposition of available caspase-6 structures underscores that zinc does not change the overall fold in caspase-6. Zinc-bound caspase-6 (4FXO, light blue) is in the helical conformation seen in unliganded, mature caspase-6 (3K7E, orange), which differs from VEID-bound (3OD5, red) caspase-6. The substrate VEID (gray spheres) indicates the substrate-binding region. Only in mature caspase-6 and the zinc-bound form of caspase-6 are the extended helical state of the 60s and 130s helices observed. C, superposition of caspase-6 active site in the apo, mature (3K7E, orange), and zinc-bound (4FXO, light blue) structures. Of the residues in this region, only His-219 undergoes a significant shift in conformation. D, superposition of VEID-bound (3OD5, red) and zinc-bound (4FXO, light blue) caspase-6 highlights the significant conformational change that caspase-6 undergoes during conversion from the helical to canonical structures. The substrate-mimic VEID (gray) is labeled as D1, I2, E3, and V4 to indicate the peptide subsite positions within the peptide.

FIGURE 4.

Anomalous difference map shows location of zinc binding in caspase-6. A, anomalous difference map calculated from data collected above the zinc absorbance edge is contoured at 5σ (purple), clearly indicating the location of zinc. The side chains and water serving as ligands are drawn as sticks. The 2F_o − F_c electron density map (blue) into which the structure was build is contoured at 1σ. B, caspase-6 dimer. The boxed regions shows active site, which contains residues appropriate for metal binding including His-121, Glu-126, and Cys-163. In this structure, these residues are not properly positioned to coordinate zinc. The caspase-6 zinc-binding exosite is shown within the side chain ligands for zinc in sticks, zinc (blue), and the water molecule (red).

FIGURE 5.

Caspase-6 exosite is not conserved. A, structure-based sequence alignment of the apoptotic human caspases in the regions of the zinc-binding sites. The active site is conserved but the caspase-6 or -9 exosites are not conserved across the apoptotic caspases. Alignments were performed structurally, and residues are numbered according to the numbering of each individual caspase. The presence of insertions and deletions in the sequences of various caspases explains why the intervals between the sites differ. B, caspase-6 in the canonical conformation shows the active site and zinc-binding exosites circled. Zinc-binding ligands are in sticks and the zincs are shown as gray spheres. The caspase-6 zinc-binding site is shown as in the crystal structure. The zinc-bound conformations of the active site and the caspase-9 exosites were modeled by selecting a new plausible rotomeric conformation for the zinc-liganding side chains and modeling a zinc molecule with proper distance for zinc binding. C, K_i value for wild-type caspase-6 inhibition by zinc is much stronger than caspase-6 variants in which the zinc ligands are deleted, suggesting that the exosite is the primary inhibitory site for zinc. This is in contrast with caspase-9, in which there is no statistical difference in the activity when the caspase-9 exosite is ablated by the C272S substitution.

FIGURE 6.

Model for the mechanism of caspase-6 inhibition by zinc. Caspase-6 exists in equilibrium between the helical conformation and the canonical conformation. Prior to substrate binding, the equilibrium strongly favors the helical conformation. To bind substrate, caspase-6 has to reorder the ends of two elongated helices into the strands or loops observed in the canonical form that are compatible with substrate binding. In the presence of zinc, the helical form of caspase-6 binds only one zinc/monomer at the exosite and is allosterically inhibited at this exosite. In the helical conformation, the active site does not bind zinc due to the disorganization of the active site residues, which are also favorable zinc-liganding residues. When caspase-6 is in the canonical conformation, zinc can also bind at the active site so that two zincs per monomer bind and inhibit caspase-6.