Inhibitory Mechanism of Caspase-6 Phosphorylation Revealed by Crystal Structures, Molecular Dynamics Simulations, and Biochemical Assays*

Background: Caspase-6 is a drug target against neurodegenerative diseases and is suppressed by phosphorylation at Ser257. Results: S257E mutation inhibits caspase-6 activation by locking the protein in the “inhibited state” and inhibits caspase-6 activity by steric hindrance. Conclusion: Phosphorylation inhibits caspase-6 through the same mechanism. Significance: The study revealed the inhibition mechanism of caspase-6 phosphorylation and provided new strategies for drug discovery. 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 Ser257 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 Ser257 inhibited self-activation of CASP6 zymogen by “locking” the enzyme in the TEVD193-bound “inhibited state.” The structural and biochemical results also showed that phosphorylation on Ser257 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.

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 ⌬proCASP6S257E 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.
Caspases, a family of cysteine proteases that cleave substrates after an aspartate residue, are the major executioners of apoptosis and inflammation and are classified into inflammatory caspases, apoptotic initiators, and effectors (1,2). CASP6 3 is classified to be an apoptotic effector by sequence similarity, and it is often activated by CASP3 during apoptosis (3). However, CASP6 also undergoes in vitro and in vivo self-activation (4,5). CASP6 is expressed as the zymogen of a short pro-domain, a large subunit (p20), an intersubunit linker (L), and a small subunit (p10), and is activated by proteolytic processing at either or both intersubunit cleavage sites (4).
Increasing evidence has demonstrated the critical roles of CASP6 in neurodegenerative diseases, including Huntington disease (HD) and Alzheimer disease (AD) (for review, see Ref. 6). In vivo proteolytic cleavage of murine htt at the CASP6 cleavage site is a crucial and rate-limiting event in the pathogenesis of HD (7). Activation of CASP6 is observed before the onset of motor abnormalities in human and murine HD brain, and active CASP6 levels correlate directly with the CAG size and inversely with the age of onset (8). Active CASP6 is abundant in the neuropathological lesions of AD (9,10). Furthermore, CASP6 mediates axon pruning through the death receptor 6 pathway during neural development (11), and many cytoskeletal proteins are targets of CASP6 (12), suggesting the importance of CASP6 in neurodegenerative processes.
Phosphorylation is critically important to caspase regulation (13). CASP6 is negatively regulated by AMP-activated protein kinase-related kinase 5 (ARK5, also known as NUAK1)-mediated phosphorylation at Ser 257 (14), but the mechanism of suppression remains unclear. CASP6 is not inhibited by IAPs (inhibitors of apoptosis) (1,2), and the alternatively spliced transcript product CASP6␤ only inhibits the activation of pro-CASP6, but not the activated CASP6 (15). So far, phosphorylation is the most efficient known inhibition mechanism for CASP6 because it inhibits both the activation and activity of CASP6 (14).
Several crystal structures of zymogen, free active and inhibitor-bound CASP6 have been published (5, 16 -19). Our previ-ous work has shown that the intersubunit cleavage site TEVD 193 binds in the active site in CASP6 zymogen and revealed a unique self-activation mechanism for CASP6 through intramolecular cleavage at Asp 193 (5). However, none of the above studies could explain the inhibitory effect of CASP6 phosphorylation. In this work, we used a CASP6 mutant to mimic the phosphorylated CASP6 and revealed the inhibition mechanism of CASP6 phosphorylation. We also used molecular dynamics (MD) simulations to test the effects of Ser 257 mutation and phosphorylation on the structure of CASP6 zymogen, and the results showed that the mutant used in this study is a good mimic of CASP6 phosphorylation.

EXPERIMENTAL PROCEDURES
Mutagenesis of CASP6-The ⌬proCASP6S257E and other mutants were generated by overlapping PCR, using WT CASP6 or ⌬proCASP6S257E as template. The DVVD 179 NQ sites of D179Th mutants were replaced by the thrombin cleavage site, LVPR 179 GS.
Protein Preparation-All CASP6 mutants were cloned in pET21b vector with a C-terminal (His) 6 tag and expressed in Escherichia coli Rosetta (DE3) strain at 18°C for 20 h. Bacterial cells harboring CASP6 mutants were cultured in LB medium supplemented with 100 g/ml ampicillin and 34 g/ml chloramphenicol at 37°C to an A 600 of 0.6 -0.8. Protein expression was induced with 0.5 mM isopropyl ␤-D-1-thiogalactopyranoside at 18°C for 20 h. Cells were harvested and resuspended in 20 mM Tris-HCl, pH 7.5, 500 mM NaCl, and 50 mM imidazole (equilibration buffer). Cells were sonicated and centrifuged at 50,000 ϫ g for 60 min at 4°C, and soluble fractions were loaded onto a 5-ml HisTrap HP column (GE Healthcare) equilibrated with equilibration buffer. The columns were washed with equilibration buffer containing 100 mM imidazole, and the target proteins were eluted with equilibration buffer containing 500 mM imidazole. ⌬proCASP6S257E proteins used for crystallization were further purified by gel filtration (120-ml Superdex-75; GE Healthcare) in buffer containing 20 mM Tris-HCl, pH 7.5, and 150 mM NaCl, and 10 mM dithiothreitol (DTT, final concentration) was added after gel filtration. To obtain fully processed ⌬proCASP6S257E, 5 g/l purified ⌬proCASP6S257E protein was incubated with 5 ng/l active CASP3 at 4°C for 2 days. Other CASP6 mutants used for biochemical analysis were purified by nickel-chelating column as described above and transferred to the same final buffer as ⌬proCASP6S257E by desalting column (5-ml HiTrap desalting column; GE Healthcare).
Crystallization and Data Collection-Both crystals of ⌬proCASP6S257E and p20/p10S257E were grown using the sitting-drop diffusion method. Crystals of ⌬proCASP6S257E were obtained by incubating 10 mg/ml protein (in 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 10 mM DTT) in 0.1 M MES, pH 6.6, 0.2 M sodium chloride, and 10% m/v polyethylene glycol (PEG) 3350 at 20°C. Crystallization solution containing 10% dimethyl sulfoxide was used for cryoprotection. The crystal was flash-frozen and maintained at 100 K by nitrogen gas during data collection. Diffraction data were collected at a wavelength of 0.98 Å on the Beamline BL5A at the KEK, Photon Factory, Tsukuba, Japan. The data were processed by the program XDS (20). The crystals belong to space group P6 5 22. Crystals of p20/p10S257E were obtained by incubating 2 mg/ml protein (in 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 10 mM DTT) in 20 mM HEPES, pH 7.5, 2.0 M NaCl at 20°C. Crystallization solution containing 10% glycerol was used for cryoprotection. The crystal was flash-frozen and maintained at 100 K by nitrogen gas during data collection. Diffraction data were collected at a wavelength of 0.98 Å on the Beamline BL17U at the SSRF (Shanghai Synchrotron Radiation facility), Shanghai, China. The data were processed by HKL2000 (21). The crystals belong to space group P2 1 .
Structure Determination and Refinement-Both structures were determined by molecular replacement calculations with PHENIX AutoMR (22) using ⌬proCASP6C163A dimer (Protein Data Bank ID code 3NR2) as the search model. The models were completed with the graphics program Coot (23) and refined using PHENIX (22). The refinement statistics are summarized in Table 1. The PDB access code for ⌬proCASP6S257E is 3V6L and for p20/p10S257E is 3V6M. The data processing and refinement statistics are summarized in Table 1.
MD Simulation of CASP6-The SANDER and PMEMD modules of AMBER 10.0 package (24) were utilized in simulations in conjunction with the AMBER ff03 all-atom force field (25) in an NVT ensemble. All MD simulations were performed as explicit solvation with approximately 3500 SPC/E waters (26). All MD simulations employed an integral time step of 2 fs, restraint of all hydrogen-containing bonds through the SHAKE algorithm (27), a cutoff of 10 Å for nonbonded interactions, and particle mesh Ewald treatment of long range electrostatic interactions (28). Because in the experimental structure, ⌬proCASP6S257E, parts of the L2 and L4 loops are flexible and their electron density is missing, we first used the SWISS-MODEL server (29) to build the missing parts. To obtain a relaxed loop structure, a 2-ns MD simulation at 600 K with a constrained protein structure except for the L2 loop was performed. Equilibration simulation at 300 K was then performed starting from the resulting structure. The simulation temperature was maintained at 300 K using Langevin dynamics with a friction coefficient of 5 ps Ϫ1 .
Purified ⌬proCASP6S257E and other mutants were diluted to 0.2 g/l in the assay buffer containing 2 g/l bovine serum albumin (BSA) and incubated at 37°C for 14 h. Samples were separated by 15% SDS-PAGE, transferred to Immobilon-P polyvinylidene fluoride (PVDF) membranes (Millipore), and probed with 1:40,000 dilution of rabbit anti-CASP6 serum, 1:5,000 dilution of secondary anti-rabbit IgG-HRP (MBL), and detected with Metal Enhanced DAB Substrate kit (Thermo Scientific).
Proteolytic Processing of ⌬proCASP6D179Th Variants by Thrombin-Purified ⌬proCASP6D179Th variants were diluted to 0.5 g/l and incubated with 5 nanounits/l thrombin (GE Healthcare) in the assay buffer at 25°C for 24 h. Samples were analyzed by 15% SDS-PAGE.
Enzyme Activity Analysis of Processed CASP6 Variants-To obtain processed CASP6 variants, 0.5 g/l purified proteins were incubated with 0.5 ng/l active CASP3 in the assay buffer at 25°C for 14 h. Protein solutions were sampled and analyzed by 15% SDS-PAGE. The activities of processed proteins were assessed by fluorogenic assay using Ac-Val-Glu-Ile-Asp-7amino-4-trifluoro-methylcoumarin (Ac-VEID-AFC) as substrate. The assay was performed with 50 M Ac-VEID-AFC in a 96-well plate with a reaction volume of 100 l, and each well contained 50 ng of processed proteins and 500 ng of BSA in the assay buffer. The activities were measured by infinite M200 multimode microplate reader (TECAN) with wavelengths of 400 nm for excitation and 505 nm for emission. The results were read at intervals of 1 min for up to 60 min. Fluorescence units were converted to the amount of moles of AFC released based on a standard curve of 0 -20 M free AFC. Cleavage rates were calculated from the linear phase of the assay.

RESULTS
To investigate the phosphorylation regulation mechanism of CASP6, Ser 257 was mutated to glutamate to mimic the phosphorylated serine residue. The resulting mutant, pro CASP6S257E, was only self-cleaved at Asp 23 , not at Asp 193 or Asp 179 (supplemental Fig. S1A), suggesting that Ser 257 phosphorylation inhibits CASP6 self-activation. The pro-domain deleted mutant ⌬proCASP6S257E did not undergo self-activation even after 15 days of incubation at 25°C (supplemental Fig. S1B) and was used for crystallization. The structure was determined at 2.2 Å and refined to R factors R work /R free of 16.9%/20.2%. The data collection and statistics from crystallographic analysis are summarized in Table 1.
Structure Analyses of ⌬proCASP6S257E-⌬proCASP6S257E exists as homodimer with each monomer assembled into a central six-stranded ␤-sheet flanked by five ␣-helices and two small ␤-strands, with four loops (L1-L4) protruding from the central ␤-sheet, forming the active site (Fig. 1A). The structure of ⌬proCASP6S257E is almost identical to CASP6 zymogen (5), with root mean square deviations of 0.41 Å for all aligned C␣ atoms (Fig. 1B). The intersubunit cleavage site TEVD 193 also binds in the active site as a ␤-strand (␤ TEVD ) in the ⌬proCASP6S257E. The ␤ TEVD containing ITEVDAA 195 forms an anti-parallel ␤-sheet with YYSHRET 222 of the L3 loop through six main chain hydrogen bonds (Fig. 1C). Superimposing the active sites of ⌬proCASP6S257E and the zymogen, we found that residues forming the substrate binding pockets and TEVD 193 both overlapped very well (Fig. 1D). The mutation site, S257E, is at the end of the helix connecting to the L4 loop. The Glu 257 forms a "salt bridge" with Lys 273 (Fig. 1E). By contrast, in CASP6 zymogen, Ser 257 cannot stabilize Lys 273 , and the side chain of Lys 273 is flexible without intact electron density ( Fig. 1F and supplemental Fig. S2). These observations indicate that the structures of ⌬proCASP6S257E and the zymogen are in the same conformation. The fact that ⌬proCASP6S257E has the intact catalytic dyad Cys 163 /His 121 but cannot undergo selfcleavage suggests that both the two structures are in the "inhibited state." However, in the zymogen, certain conformational changes can trigger the intramolecular self-cleavage, whereas in ⌬proCASP6S257E, the single mutation "locks" the protein in the inhibited state, and the salt bridge between Glu 257 and Lys 273 may play a key role for the locking mechanism.
Compared with the Ac-VEID-CHO bound CASP6 (representing fully active enzyme, in the "active state"), in ⌬proCASP6S257E, Asp 193 does not fully bind in the S1 pocket (constructed by Arg 64 , Asn 161 , and Arg 220 ; Fig. 2A). In the Ac-VEID-CHO-bound CASP6, the Arg 64 and Arg 220 are engaged in strong salt bridges with the P1 aspartate, which is further hydrogen-bonded to Asn 161 (Fig. 2B). The carbonyl group of the P1 aspartate covalently bonds to sulfur of Cys 163 , and the carbonyl oxygen is stabilized in the "oxyanion hole" formed by the main chain amino nitrogen of Cys 163 and imidazolyl nitrogen of His 121 . In contrast, in ⌬proCASP6S257E, the side chain carboxyl oxygen of Asp 193 is approximately 5 Å from the guanidinium group of Arg 220 and even farther away from Arg 64 and Asn 161 . The distance between the sulfur of Cys 163 and the main chain carbonyl of Asp 193 is approximately 4.5 Å, and the main chain carbonyl oxygen of Asp 193 points to the opposite direction of the oxyanion hole. At this conformation, it is hard for Cys 163 to nucleophilically attack the main chain carbonyl of Asp 193 and to initiate the self-cleavage. Furthermore, the L3 loops of the two structures deviate at His 219 , and the main chain amino nitrogen of Tyr 217 split about 2.9 Å (Fig. 2C). In ⌬proCASP6S257E, the six hydrogen bonds between TEVD 193 and the L3 loop fasten this cleavage site on the L3 loop, and therefore the conformation of TEVD 193 varies with the conformation of the L3 loop. The phenolic group of Try 217 hydropho- bically interacts with the side chain of Lys 272 , and the imidazolyl nitrogen of His 219 and the main chain amino nitrogen of Tyr 217 form two hydrogen bonds with the main chain ketonic oxygens of Lys 272 and Lys 273 , respectively (Fig. 2D). These interactions lead to the coupling between the L3 and L4 loops. More importantly, Glu 257 forms a salt bridge with Lys 273 , which stabilizes the side chain of Lys 273 and further locks TEVD 193 in the inhibited state conformation. In Ac-VEID-CHO-bound CASP6, His 168 of the L2 loop forms a hydrogen bond with His 219 , and with the insertion of His 168 between the L3 and L4 loops, these two loops do not directly interact. These observations indicate that after cleavage at Asp 179 , the conformational changes of the L2 loop could break the interaction network between the L3 and L4 loops and release the CASP6 from the inhibited state.
MD Simulations Show Good Resemblance between S257E Mutation and Phosphorylation-To test how well the S257E mutation resembles phosphorylation and to gain molecular detailed understanding of the effects of phosphorylation on the activation of CASP6, we performed MD simulations using the ⌬proCASP6S257E crystal structure, the structure with the mutation of Glu 257 to phosphorylated serine (representing the phosphorylated CASP6 zymogen), and the structure with Glu 257 mutated back to serine (representing the WT CASP6 zymogen), respectively. The simulation results showed that the fluctuation of the distance between the sulfur of Cys 163 and the main chain carbonyl of Asp 193 in ⌬proCASP6S257E or phosphorylated CASP6 zymogen is smaller than that in WT CASP6 zymogen (Fig. 3). The results indicated that both S257E mutation and phosphorylation have similar effects on freezing the motions of the active site, and the active site of the WT zymogen is much more flexible. The results suggested that S257E mutation is a good mimic of Ser 257 phosphorylation and further confirmed that the self-activation of CASP6 zymogen is inhibited through the locking mechanism.
Biochemical Assays Agreed with Structure Analyses-To determine further the specific contribution of each residue, the key residues involved in the interaction network around Glu 257 were mutated to alanine separately. Purified ⌬proCASP6S257E and ⌬proCASP6(S257E,H219A) proteins did not self-activate ( Fig. 4, A and B). By contrast, ⌬proCASP6(S257E,Y217A) and ⌬proCASP6(S257E,K272A) already contained little selfcleaved enzyme before incubation, indicating that these two mutants underwent self-activation during purification. These results demonstrated that CASP6S257E regained self-activation ability by breaking the hydrophobic interaction between Tyr 217 and Lys 272 and indicated that this hydrophobic interaction was essential in constructing the network. Furthermore, to disrupt the salt bridge between Glu 257 and Lys 273 , Lys 273 was mutated to alanine, or Glu 257 was mutated to lysine or glutamine. The resulting ⌬proCASP6(S257E,K273A), ⌬proCASP6S257K, and ⌬proCASP6S257Q also regained selfactivation ability, which confirmed the key role of salt bridge between Glu 257 and Lys 273 in the inhibition mechanism.
To investigate the influence of cleavage at DVVD 179 on the phosphorylation-inhibited CASP6, DVVD 179 NQ was replaced by the thrombin cleavage site, LVPR 179 GS, and the resulting mutants will be referred to as D179Th. These mutants were cleaved at their thrombin sites and transferred to their active form, p20/Lp10, by incubating with thrombin. Thrombin could not cleave the TEVD 193 site, which was evidence that  thrombin did not cleave ⌬proCASP6(S257E,D179Th,C163A) at TEVD 193 (Fig. 4C). Notably, the TEVD 193 site of ⌬proCASP6(S257E,D179Th) was cleaved, as the p10 band appearing after the D179Th site was cleaved (Fig. 4D), indicating that ⌬proCASP6S257E could self-cleave at TEVD 193 after the 179 site was cleaved.
Structure analyses suggest that after cleavage at Asp 179 , the rotation of the L2 loop results in the insertion of His 168 between the L3 and L4 loops and breaking their interaction network. Indeed, after cleavage at the D179Th site, ⌬proCASP6(S257E,D179Th,H168A) and ⌬proCASP6(S257 E,D179Th,H219A) (Fig. 4, E and F) self-cleaved at Asp 193 much slower than ⌬proCASP6(S257E,D179Th), indicating that the formation of the hydrogen bond between His 168 and His 219 was crucial to the network disruption. By contrast, ⌬proCASP6 (S257E,D179Th,Y217A) and ⌬proCASP6(S257E,D179Th,K2 72A) self-cleaved at Asp 193 much faster (Fig. 4, G and H), further confirming the dominant role of the hydrophobic interaction between Tyr 217 and Lys 272 in constructing the network. The quantitative data of thrombin proteolytic processing assay (Fig. 4, C-H) are summarized in supplemental Table S1.
These results all agree with the conclusions drawn from the structural analyses which showed that CASP6 is locked in the inhibited state through the interaction network among TEVD 193 , the L3 and the L4 loops, as well as the salt bridge between Glu 257 and Lys 273 . Meanwhile, the cleavage at Asp 179 followed by insertion of His 168 between the L3 and L4 loops breaks the interaction network, and further releases CASP6 from the inhibited state.
S257E Inhibits Activity of Cleaved CASP6 by Steric Hindrance-To investigate whether phosphorylation inhibits the activity of activated CASP6, ⌬proCASP6S257E was processed by 0.1% active CASP3 for 14 h (supplemental Fig.  S3A). The activity of the processed protein (referred as p20/ p10S257E in the following discussion) was approximately 0.4% of that of WT CASP6 and was one third that of proCASP6D (23,179,193)A (representing the activity of CASP6 zymogen, supplemental Fig. S3B). The low activity of p20/p10S257E suggests that phosphorylation at Ser 257 also inhibits the activity of activated CASP6. Furthermore, the activities of CASP3-processed ⌬proCASP6 (S257E,Y217A), ⌬proCASP6(S257E,K272A), and ⌬pro-CASP6(S257E,K273A) were as low as ⌬proCASP6S257E (supplemental Fig. S3B), indicating the mutations that regained CASP6 self-activation ability cannot increase the activity of CASP6S257E and suggesting that phosphorylation inhibited the activity of processed CASP6 through a different mechanism.
The p20/p10S257E was crystallized for structure determination. The crystals belong to space group P2 1 , with four (p20/ p10) 2 dimers in one asymmetric unit. The four CASP6 dimers in the same asymmetric unit are almost identical, with root mean square deviations of between 0.11 and 0.22 Å for all aligned C␣ atoms. For the following discussion, we will only refer to the A/B dimer. The structure of p20/p10S257E is very similar to that of free active CASP6 (18), with typical caspase ␣/␤-fold, but most parts of the loops forming the active sites are flexible without electron density, namely residues 164 -179, 194 -197 of L2, residues 213-221 of L3, and residues 260 -274 of L4 (Fig. 5A). In the free active CASP6, the N terminus of p10 is oriented toward the dimer interface (18), and substrate binding induces the N terminus of p10 to rotate 180°and form a loop bundle with the L2 and L4 loops as observed in the Ac-VEID-CHO-bound CASP6. Superimposing the structures of p20/p10S257E and Ac-VEID-CHO bound CASP6, we found that the loop bundle is well formed in the inhibitor-bound CASP6; however, the mutation site Glu 257 of p20/p10S257E overlapping with the L2Ј loop of inhibitor bound CASP6 (Fig. 5B). These observations indicate that with the S257E mutation, the loop bundle cannot be formed because of steric hindrance, which agrees well with the low activity of p20/p10S257E.
To prove this hypothesis, Ser 257 was mutated to 17 other amino acids except for proline and glycine, and the mutants were processed by CASP3. All of the mutants with small side chains, including S257A, S257V, S257C,S257T, and S257I, had a relatively high activity, comparable with WT CASP6; whereas all of the mutants with big side chains, including S257E, S257D, S257Q, S257N, S257F, S257Y, S257W, S257K, S257R, S257L, and S257H had a relatively low activity, Ͻ3% activity of the WT CASP6, and comparable with proCASP6D (23,179,193)A (Fig.  5C). The mutant S257M was the only exception; it has a long side chain, but its activity was relatively high, which may be due to the side chain flexibility of methionine. These results are consistent with the notion that the activities of CASP6 mutants depended on the side chain size of the 257 residue, confirming that phosphorylation inhibited CASP6 activity by steric hindrance.

DISCUSSION
In this work, we used the S257E mutation to mimic the Ser 257 -phosphorylated CASP6. Our structural and biochemical results revealed the S257E mutation inhibited self-activation of CASP6 zymogen and the activity of processed CASP6 through two different mechanisms. It inhibited selfactivation of CASP6 zymogen by locking the CASP6 in the TEVD 193 -bound inhibited state. The salt bridge between Glu 257 and Lys 273 fixed the position of Lys 273 and further locked the zymogen in the inhibited state through the interaction network among TEVD 193 , the L3 loop, and the L4 loop. It inhibited the activity of processed CASP6 by steric hindrance because the side chain of Glu 257 hindered the formation of loop bundle, which is crucial for the activities of caspases in general. Based on the fact that phosphorylated serine has the same charge and a similar size with a glutamate, we propose that Ser 257 phosphorylation inhibits CASP6 through the same mechanism as S257E mutation. This proposal was further validated by MD simulations, which showed that both the S257E mutant and the WT CASP6 with the phosphorylated Ser 257 show similar structural and dynamical properties. Compared with the unphosphorylated WT protein, the active site in both protein forms became less flexible and prevented the close approach between the Cys 163 and the Asp 193 , which is essential for the self-activation of the enzyme. ARK5 inhibits CASP6 activation by phosphorylation at Ser 257 in vitro and in vivo, and this phosphorylation also suppresses the CASP3-mediated activation of CASP6 (14). The inhibitory mechanism of CASP6 phosphorylation revealed by our structural, biochemical, and MD simulation results agreed very well with those early studies.
This study has extended our understanding of the molecular mechanism of CASP6 activation and regulation. Our previous study revealed that CASP6 could undergo self-activation through intramolecular cleavage at Asp 193 , and we have proposed that the ordered TEVD 193 conformation has both activation and inhibition function in regulating CASP6 activity (5). The presence of TEVD 193 in the active site of zymogen inhibits the activation and activity of CASP6 by excluding other substrate binding and preventing this site from intermolecular cleavage; meanwhile, because TEVD 193 is located very close to the catalytic site, a small conformational change would be enough to induce the intramolecular self-cleavage. The phosphorylated and unphosphorylated CASP6 zymogens are in the same TEVD 193 -bound inhibited state, but they have different fates. The unphosphorylated zymogen can initiate self-activation through certain conformational change, whereas the phosphorylated zymogen is locked in the inhibited state. These results further suggest that dephosphorylation by a specific phosphatase may be a trigger to the intramolecular self-cleavage.
Crucial roles for CASP6 in HD and AD have been well documented (6), and regulating the activation and activity of CASP6 could be an efficient method to develop pharmaceutical therapy against these neurodegenerative disorders. Most of reported caspase inhibitors are peptide-based and mimic the cleavage site in caspase substrates, but these inhibitors often lack selectivity and may act on more than one caspase target (30). Moreover, peptides have poor cell penetration properties and are unlikely to fulfill criteria for bioavailability after oral uptake (31). Therefore, specific and effective inhibitors are still in demand. Nonpeptide inhibitors have only recently been described for CASP6 (32,33), but these inhibitors also target the active site of the enzyme. ARK5 inhibits both activation and activity of CASP6 by phosphorylation, and it regulates the expression level of CASP6 indirectly because it phosphorylates and suppresses p53 (34), and CASP6 is directly transactivated by p53 (35). These functions make AKR5 a potential therapeutic target. Meanwhile, because phosphorylation inhibits both self-activation of CASP6 and the activity of processed CASP6, and this phosphorylation site is unique for CASP6 (13,14), phosphorylating CASP6 or mimicking the phosphorylation state may lead to a novel strategy for drug discovery rather than directly targeting the active site. Therefore, our results not only revealed the molecular mechanism of this inhibition but also provided the essential structural basis for rational drug design outside the active site.