Dissecting an allosteric switch in caspase-7 using chemical and mutational probes

Abstract

Apoptotic caspases, such as caspase-7, are stored as inactive protease zymogens, and when activated, lead to a fate-determining switch to induce cell death. We previously discovered small molecule thiol-containing inhibitors that when tethered revealed an allosteric site and trapped a conformation similar to the zymogen form of the enzyme. We noted three structural transitions that the compounds induced: (i) breaking of an interaction between Tyr-223 and Arg-187 in the allosteric site, which prevents proper ordering of the catalytic cysteine; (ii) pinning the L2' loop over the allosteric site, which blocks critical interactions for proper ordering of the substrate-binding groove; and (iii) a hinge-like rotation at Gly-188 positioned after the catalytic Cys-186 and Arg-187. Here we report a systematic mutational analysis of these regions to dissect their functional importance to mediate the allosteric transition induced by these compounds. Mutating the hinge Gly-188 to the restrictive proline causes a massive approximately 6000-fold reduction in catalytic efficiency. Mutations in the Arg-187-Tyr-223 couple have a far less dramatic effect (3-20-fold reductions). Interestingly, although the allosteric couple mutants still allow binding and allosteric inhibition, they partially relieve the mutual exclusivity of binding between inhibitors at the active and allosteric sites. These data highlight a small set of residues critical for mediating the transition from active to inactive zymogen-like states.

FIGURE 1.

Allosteric site and dimeric structure in caspase-7. A, the surface of active site-bound caspase-7 shows a large open allosteric (yellow) site at the dimer interface. This cavity is distinct from the active sites, which are bound with the active site inhibitor DEVD (green sticks). B, large subunits of caspase-7 dimers (dark green and dark purple) contain the active site cysteine-histidine dyad. The small subunits (light green and light purple) contain the allosteric site cysteine 290. The conformation of the substrate-binding loops (L2, L2′, L3, and L4) in active caspase-7 (Protein Data Bank (PDB) number 1f1j) is depicted. The L2′ loop (spheres) from one-half of the dimer interacts with the L2 loop from the other half of the dimer. C, binding of allosteric inhibitors influences the conformation of the L2′ loop (spheres), which folds over the allosteric cavity (PDB number 1shj). Subunit rendering is as in panel A. Panels A, B, and C are in the same orientation.

FIGURE 2.

Structure of allosteric inhibitors DICA and FICA. DICA and FICA are hydrophobic small molecules that bind to an allosteric site at the dimer interface of caspase-7. Binding of DICA/FICA is mediated by a disulfide between the compound thiol and Cys-290 in caspase-7.

FIGURE 3.

Movement of L2′ blocking arm. The region of caspase-7 encompassing the allosteric couple Arg-187 and Tyr-223 is boxed. The inset shows the down orientation of Arg-187 and Tyr-223 in the active conformation with DEVD substrate mimic (orange spheres) in the active site. In the allosteric/zymogen conformation, Arg-187 and Tyr-223 are pushed up by DICA (blue spheres).

FIGURE 4.

Rational design of mutations in the caspase-7 allosteric couple. Interrogated residues are shown in stick representation to clarify the steric relationship between these residues. Allosteric couple residues Arg-187 and Tyr-223 and catalytic dyad residues His-144 and Cys-186 are drawn in cyan sticks. Allosteric inhibitor DICA is in orange sticks, and the cysteine residue to which it covalently binds (Cys-290) is marked. Residue Gly-188 is in purple sticks, and Tyr-229 is in yellow sticks. This figure is based on the allosterically inhibited complex of caspase-7 with DICA (PDB number 1shj).

FIGURE 5.

Monitoring mutual exclusivity of binding at the active and allosteric sites of caspase-7 variants. A, the proportion of caspase-7 small subunit bound by DICA and large subunits bound by DEVD are shown. Caspase-7 variants were first incubated with the allosteric inhibitor DICA (gray bars) and then incubated with active site inhibitor DEVD-fmk (black bars). Binding of DICA to Cys-290 in the small subunits and binding of DEVD to the active site cysteine in the large subunits was monitored by mass spectrometry. WT, wild type. B, as in A, but caspase-7 variants were first incubated the allosteric inhibitor FICA (white bars) and then with active site inhibitor DEVD-fmk (black bars).

FIGURE 6.

Effect of order of addition on mutual exclusivity of active and allosteric sites. A, caspase-7 variants were first incubated with active site inhibitor DEVD-fmk (black bars) and then with the allosteric inhibitor DICA (gray bars). Binding of DICA to Cys-290 in the small subunits and binding of DEVD to the active site cysteine in the large subunits was monitored by mass spectrometry. WT, wild type. B, as in A, but caspase-7 variants were first incubated with active site inhibitor DEVD-fmk (black bars) and then with the allosteric inhibitor FICA (white bars).

FIGURE 7.

Model of conformational equilibrium of wild type and allosteric-couple mutations in caspase-7. The conformational equilibrium in the presence of an active site binder lies stringently toward the active conformation. The conformational equilibrium when bound to the allosteric inhibitor lies less stringently toward the allosteric/zymogen state, as indicated by the size of the arrows.