Inhibitory mechanism of caspase-6 phosphorylation revealed by crystal structures, molecular dynamics simulations, and biochemical assays

Abstract

The apoptotic effector caspase-6 (CASP6) has been clearly identified as a drug target due to its strong association with neurodegeneration and axonal pruning events as well as its crucial roles in Huntington disease and Alzheimer disease. CASP6 activity is suppressed by ARK5-mediated phosphorylation at Ser(257) with an unclear mechanism. In this work, we solved crystal structures of ΔproCASP6S257E and p20/p10S257E, which mimicked the phosphorylated CASP6 zymogen and activated CASP6, respectively. The structural investigation combined with extensive biochemical assay and molecular dynamics simulation studies revealed that phosphorylation on Ser(257) inhibited self-activation of CASP6 zymogen by "locking" the enzyme in the TEVD(193)-bound "inhibited state." The structural and biochemical results also showed that phosphorylation on Ser(257) inhibited the CASP6 activity by steric hindrance. These results disclosed the inhibition mechanism of CASP6 phosphorylation and laid the foundation for a new strategy of rational CASP6 drug design.

FIGURE 1.

Structure of ΔproCASP6S257E. A, overall structure of ΔproCASP6S257E. B, structure overlay of ΔproCASP6S257E and CASP6 zymogen. C, active sites of ΔproCASP6S257E. D, active sites overlay of ΔproCASP6S257E and CASP6 zymogen. E and F, residues surrounding 257 site in ΔproCASP6S257E (E) and in structure overlay of ΔproCASP6S257E and CASP6 zymogen (F). The electron density map (2F_o − F_c maps) was shown at 1.0 σ, calculated by PHENIX.refine. The salt bridge and hydrogen bonds are represented by black dashed lines.

FIGURE 2.

Active sites overlay of ΔproCASP6S257E and Ac-VEID-CHO bound CASP6. A–D, structure superimposition of whole active sites (A), substrates and S1 pockets (B), L3 loops, the S1 pockets, and the Cys/His dyads (C), and L3–L4 loop interaction (D). The black dashed lines represent the hydrogen bonds, and red dashed lines show the distance between two atoms.

FIGURE 3.

MD simulation of ΔproCASP6S257E, phosphorylated CASP6 zymogen, and WT CASP6 zymogen. The distance between the sulfur of Cys¹⁶³ and main chain carbonyl of Asp¹⁹³ in three different mutant/state of CASP6 zymogen were calculated by MD simulation and are shown on the histogram as a function of time. S257Sp, Ser²⁵⁷ mutated to phosphorylated serine.

FIGURE 4.

A and B, self-activation analysis of CASP6 variants from 0 (A) to 14 h (B) by Western blotting. The asterisk-labeled bands were a bacterial contamination protein. C–H, Coomassie Blue-stained SDS-polyacrylamide gels of ΔproCasp6S257E,D179Th,C163A (C), ΔproCasp6S257E,D179Th (D), ΔproCasp6S257E,D179Th,H168A (E), ΔproCasp6S257E,D179Th,H219A (F), ΔproCasp6S257E,D179TH,Y217A (G), and ΔproCasp6S257E,D179Th,K272A (H) incubated with thrombin for 24 h.

FIGURE 5.

S257E inhibits the activity of cleaved CASP6 by steric hindrance. A, overall structure of p20/p10S257E. B, superimposition of loop bundle region in Ac-VEID-CHO-bound CASP6 and p20/p10S257E. The hydrogen bonds are represented by black dashed lines, and Glu/Ser²⁵⁷ are shown as spheres. C, VEIDase activity of CASP6 variants. The activities of processed CASP6 variants were normalized to WT CASP6. The activities with low values are also shown in a histogram with appropriate maximum scale of vertical axis. Assays were done in triplicate, and error bars represent S.D. The activity of 1/1000 CASP3 is shown as a negative control. The processed CASP6 variants were analyzed by SDS-PAGE shown below the histogram.