Bioorganic & Medicinal Chemistry Letters xxx (2011) xxx–xxx

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Bioorganic & Medicinal Chemistry Letters

journal homepage: www.elsevier .com/ locate/bmcl

Structure of human caspase-6 in complex with Z-VAD-FMK: New peptidebinding mode observed for the non-canonical caspase conformation

Ilka Müller a, Marieke B. A. C. Lamers a, Alison J. Ritchie a, Celia Dominguez b,Ignacio Munoz-Sanjuan b, Alex Kiselyov b,⇑a BioFocus, Chesterford Research Park, Saffron Walden, Essex, CB10 1XL, UKb CHDI Foundation, Inc., 6080 Center Drive, Suite 100, Los Angeles, CA 90045, USA

a r t i c l e i n f o a b s t r a c t

Article history:Received 27 May 2011Revised 7 July 2011Accepted 11 July 2011Available online xxxx

Keywords:ApoptosisHuntington’s DiseaseHuman caspase-6Irreversible inhibitor bindingStructure-based drug design

0960-894X/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.bmcl.2011.07.041

⇑ Corresponding author.E-mail address: alex.kiselyov@chdifoundation.org

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Caspase-6 is a cysteine protease implicated in neuronal survival and apoptosis. Deregulation of caspase-6activity was linked to several neurodegenerative disorders including Alzheimer’s and Huntington’s Dis-eases. Several recent studies on the structure of caspase-6 feature the caspase-6 zymogen, mature apo-caspase-6 as well as the Ac-VEID-CHO peptide complex. All structures share the same typical dimeric cas-pase conformation. However, mature apo-caspase-6 crystallized at low pH revealed a novel, non-canon-ical inactive caspase conformation speculated to represent a latent state of the enzyme suitable for thedesign of allosteric inhibitors. In this treatise we present the structure of caspase-6 in the non-canonicalinactive enzyme conformation bound to the irreversible inhibitor Z-VAD-FMK. The complex features aunique peptide binding mode not observed previously.

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Caspases are cysteine proteases that cleave their target after anaspartate residue and are involved in cellular processes such as celldifferentiation or aging. Caspase deregulation has been associatedwith cancer, neuromuscular and neurodegenerative disorders.Deregulation of caspase-6 activity plays an important role in dis-eases like Alzheimer’s or Huntington’s Disease (HD).1,2 In HD, a mu-tant huntingtin (mHTT) protein features expanded polyglutaminerepeats at its N-terminus. Caspase-6 is a key processing enzymeresponsible for the cleavage of mHTT. The cleavage generates frag-ments that contribute to the HD pathology3,4, and inhibition ofcaspase-6 activity may prove to be neuroprotective. Caspase-6 isexpressed as a pro-enzyme with a 23 amino acid pro-domain andan inter domain linker; both are removed via a caspase-3 mediatedprocessing upon maturation to yield a large N-terminal (p20) andsmaller C-terminal (p10) domain. Structural studies on differentforms of caspase-6 have been a focus of several publications. Forexample, the caspase-6 zymogen with the inter domain linker at-tached, the mature apo-caspase-6 and the caspase-6 Ac-VEID-CHO peptide complex feature caspase-6 in a conformation similarto the one observed for the related caspases-3 and -7.5,6 Each ofthe two p20/p10 monomers of the active dimer assembles into acentral six-stranded b-sheet which is flanked by five a-helicesand three small b-strands. In the peptide complex, four loops (L1–L4) extend from the central b-sheet to form the peptide binding site.

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(A. Kiselyov).

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As suggested for other caspase-inhibitor complexes, the peptideinhibitor carbonyl functionality forms a covalent bond with the cat-alytic cysteine Cys163. The carbonyl is further hydrogen-bondingwith histidine His121, which is part of the catalytic cysteine-histi-dine diad. A subsequent study reported an alternative conformationfor mature apo-caspase-6, with several distinct features near thepeptide binding site which had not been observed for other caspas-es.7 Namely, residues 61–65 and 121–135 which in the canonicalconformation adopt loop- and b-hairpin-conformations, respec-tively, now extend two of the central a-helices. This causes the cat-alytic histidine His121 to position away from Cys163 in anorientation that does not support caspase activity. It was thereforeconcluded that the observed apo-enzyme structure represents aninactive conformation of caspase-6. We analysed the experimentalconditions of all caspase-6 crystal structures available to date forthe presence of a common determinant of the respective conforma-tion, thereby identifying the pH as a possible contributing factor:Specifically, we suggest that crystallization of caspase-6 atnear-neutral pH favors the active conformer, whereas the inactivenon-canonical form of the enzyme may be linked to acidic condi-tions. Vaidya et al. recently reported on the increased stability ofthe non-canonical form suggesting a ligand-induced transition tothe canonical conformer.8 To test whether peptide binding is suffi-cient to facilitate a non-canonical-to-canonical conformationalchange, we solved the crystal structure of caspase-6 in complexwith Z-VAD-FMK9, a nonspecific covalent caspase inhibitor, atacidic pH.

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A wide range of sparse matrix screens were run to identify suit-able crystallization conditions for caspase-6 in complex with Z-VAD-FMK. Notably, only acidic conditions yielded crystals suitablefor diffraction experiments for the Z-VAD-FMK complex.

Application of sodium citrate buffer at pH 4.3 produced crystalsdiffracting to 2.65 Å with two p202/p102 tetramers in the asym-metric unit (chains A/B/C/D and E/F/G/H, respectively, Fig. 1a andb). When the model of apo-caspase-6 at physiological pH [PDB ID3P45] was used for molecular replacement, substantial deviationsof the loop regions around the active site were apparent from theinitial electron density maps. These mismatches were attributedto a rearrangement similar to the one observed for the inactivatecaspase-6 conformation reported at pH 4.6 [PDB ID 2WDP]. Conse-quently, the low pH-model was used for molecular replacement,resulting in a significant increase of the map correlation coefficientfrom 59% to 74% compared to the earlier MR solution using the pH7.4 model. Clear peaks for the Z-FAD-FMK ligand were visible inthe initial electron density maps in three of the four p20/p10 di-mers in the asymmetric unit (Fig. 1c). The ligand density was lesswell defined in the fourth dimer; hence the ligand was notmodeled for this subunit (chain E).

The overall fold resembles the apo-caspase-6 structure attainedat pH 4.6 with an r.m.s.d. of 1.0 Å over 203 Ca atoms (Fig. 2a). It is

Figure 1. Structure of caspase-6 in complex with Z-VAD-FMK. (a): Ribbonschematic representation of the two p202/p102 tetramers in the asymmetric unitof the Z-VAD-FMK caspase-6 complex structure; chain A/B colored in blue/cyan,chain C/D colored in green/light green, chain E/F colored in grey/light grey andchain G/H colored in red/salmon, respectively. The Z-VAD-FMK peptide moleculesare highlighted as ball and stick. (b): Wire representation of caspase-6 p20/p10subunit A/B with bound Z-VAD-FK peptide (yellow). The strands of the central six-stranded b-sheet are highlighted in blue, and the flanking a-helices are highlightedin cyan. The helix extension at helix I and II, respectively, is highlighted in red.Loops constituting the peptide binding site are colored in purple (L3) and orange(L4), respectively. Loop L2’ (yellow) resides at the p10/p10 interface. (c) Final modelfor the Z-VAD-FMK site in subunit A/B and initial 2Fo–Fc (blue) and Fo–Fc (green)electron density maps contoured at 1 and 2.5r, respectively.

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distinct from the conformation observed for caspase-6 in the Ac-VEID-CHO complex [PDB ID 3OD5] exhibiting an r.m.s.d. of 4.0 Åfor 225 Ca atoms (Fig. 2b). Most significantly, residues 121–135which form a b-hairpin in the Ac-VEID-CHO complex and are alsobelieved to mediate substrate interactions in other caspases arefound to elongate one of the central a-helices (residues 136–142,Helix I) in the Z-VAD-FMK complex (Fig. 2e). Furthermore, a helixspanning residues 66–81 (Helix II) in the Ac-VEID-CHO complex isextended by an extra turn at its N-terminus in the Z-VAD-FMKcomplex. These two helix extensions lead to blocked P1 and P3peptide binding sites when compared to the caspase-6 Ac-VEID-CHO complex. As a result, the Z-VAD-FMK peptide is shifted withrespect to the L3 loop (residues 217–222) and backbone–backboneinteractions between the FMK peptide and caspase-6 are observedat Tyr216, Ser218 and His219 compared to Ser218, His219 andArg220 for the Ac-VEID-CHO complex (Fig. 3b and c). As antici-pated, the Z-VAD-FMK peptide is linked covalently to Cys163 fea-turing estimated C–S bond length of 1.82 Å. The side chain of thecatalytic histidine His121 is rotated away from Cys163 and doesnot interact with the Z-VAD-FMK peptide. The terminal benzoylmoiety of the inhibitor is positioned in a cavity formed byHis219 and Lys272 side chains on the L3 and L4 loops, respectively.Conformation of the L4 loop (residues 257 274) differs slightly be-tween the four p20/p10 dimers in the asymmetric unit. L4 loop res-idues are partly disordered in subunits E/F and G/H, but theirconformation is clearly defined by the electron density in chainsA/B and C/D. When compared to the caspase-6 Ac-VEID-CHO com-plex, the L4 loop is not folded back onto the peptide binding sitebut rather stays in a more open conformation. The L2 loop of theZ-VAD-FMK complex (residues 164–175) is disordered beyondArg164 in all four p20/p10 subunits. The L2’ loop resides at theinterface between the p10/p10’ subunits of the p202/p102 tetrameras observed for the apo-caspase-6 in both inactivated and activatedconformations ( Fig. 2a) rather than rotating out and forming abundle with L2 and L4 loop of the neighboring p20/p10 subunitas observed for the caspase-6 Ac-VEID-CHO complex (Fig. 2b).Interestingly, the Z-VAD-FMK complex of caspase-1 crystallizedat pH 6.0 [10; PDB ID 2HBQ] yields a Z-VAD-FMK peptide bindingmode similar to the one observed for Ac-VEID-CHO in the caspase-6 complex, but distinct from the one observed for Z-VAD-FMKbound to caspase-6 (Fig. 2d/g and c/f, respectively).

A recent study suggested that in solution, caspase-6 may fea-ture a latent conformation that undergoes a transition upon ligandbinding, which had not been observed for other caspases.8 Explic-itly, the results indicated enrichment in helical features for thelatent compared to the ligand bound state. Our results query thisinterpretation, since the helix extensions observed for the apo-cas-pase-6 crystallized at acidic pH are also present in the Z-VAD-FMKcaspase-6 complex (Fig. 2a).

In conclusion, our structural studies on the Z-VAD-FMK inhibi-tor complex show that the latent non-canonical conformer is alsocapable of peptide binding. Since the structure was obtained atnon-physiological pH, we caution that the binding mode mightnot be physiologically relevant and drug design based on thenon-canonical conformation may yield false positive results.Further studies on ligand binding capabilities of caspase-6 at acidicpH are warranted to probe the relevance of the observednon-canonical conformation.

Experimental procedures. Protein expression and purification:Mature human caspase-6 was expressed and purified as describedpreviously.6 In brief, zymogen-caspase-6 (amino acid residues 24–293) with a C-terminal 6 His-tag was expressed in the RosettaE. Coli expression strain. The caspase-6 zymogen underwent self-cleavage at Asp179 and Asp193 during protein expression to yield0.5 mg of the mature active apo-caspase-6 from 6 L culture. Theprotein was purified by NiNTA affinity chromatography followed

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Figure 2. Panels (a–d) show overlays of caspase-6 bound to Z-VAD-FMK (PDB ID 3QNW, the protein is displayed as wire-model with the same coloring scheme as used inFigure 1b and the peptide is displayed as stick-model with carbon atoms colored in yellow). The central core region shows only minor differences between the structures, butmajor conformational rearrangement around the ligand binding sites. Panels (e–g) focus on the ligand binding site of the peptide complexes. The central core is displayed incartoon representation based on the 3QNW (e and f) or 3OD5 (g) coordinates, respectively. The same color is used both for flexible loops and ligand carbon atoms of eachcomplex. (a): Mature apo-caspase-6 crystallized at pH 4.6 (PDB ID 2WDP) and the caspase-6 Z-VAD-FMK complex share the same overall fold, including the extended centralhelices (red). (b),(e) Overlay with caspase-6 in complex with Ac-VEID-CHO (PDB ID 3OD5, ligand carbon atoms colored in blue) shows that the inhibitor binding mode ismutually exclusive. (c),(f) and (d),(g) Overlay of caspase-1 Z-VAD-FMK complex (PDB ID 2HBQ, ligand carbon atoms colored in green) with the caspase-6 Z-VAD-FMK andcaspase-6 Ac-VEID-CHO complex, respectively. The overall topology of the two Z-VAD-FMK complexes shows large deviations between the peptide binding modes in caspase-6 and -1, respectively. The Z-VAD-FMK binding in caspase-1 closely resembles the one observed for Ac-VEID-CHO in complex with caspase-6.

Figure 3. (a) and (b) Protein-peptide interaction in the Z-VAD-FMK caspase-6 complex, the hydrogen bond network is depicted in (b) as dotted lines. The Z-VAD-FMK peptideis covalently linked to Cys163. His121 is rotated away from and not interacting with the peptide. Hydrogen bonds are observed between the backbone of the peptide and L3loop residues Tyr216, Ser218 and His219 in caspase-6. (c) In comparison, the backbone-backbone interaction between peptide and L3 loop in the caspase-6 Ac-VEID-CHOcomplex (PDB-ID 3OD5) involves residues Ser218, His219 and Arg220.

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by size exclusion chromatography. The purified protein was con-centrated and buffer exchanged on a PD-10 column against25 mM Tris pH 8.0 and 50 mM NaCl. Apo-Caspase-6 at a proteinconcentration of 0.3 mg/ml was then incubated with 100 lM Z-VAD-FMK (10 mM stock in 100% DMSO, purchased from Calbio-chem, Cat. No.: 219007) at room temperature for 2.5 h. Inhibitedprotein was further concentrated by ultrafiltration and used forcrystallization trials. The protein concentration was determinedusing Coomassie Plus reagent, measuring optical absorbance at595 nm. Ten millimolar DTT was added to the concentrated proteinprior to crystallization.

Crystallization and X-ray analysis. Factorial crystallizationscreens used for initial screening included the Molecular Dimen-sions ProPlex screen, and the Emerald BioSystems Wizard I and IIscreens in a 96-well format. Initial crystals of the caspase-6 Z-VAD-FMK complex grew in 0.1 M phosphate-citrate pH 4.2, 0.2 Mlithium sulphate and 10% v/v 2-propanol at 20 �C using a proteinconcentration of 9 mg/ml. Optimization trials were performed in

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24-well plates (VDXm from Hampton Research). The best resultswere obtained in 0.1 M lithium sulfate, 0.1 M sodium citrate pH4.3 and 11% 2-propanol.

To improve the size of the crystals, micro-seeding experimentswere performed in 24-well plates by streak seeding through fourdrops sequentially using the crystals from previous experiments.The seeding experiments were set up mixing 0.1 M lithium sulfate,0.1 M sodium citrate pH 4.3 and 11% v/v 2-propanol and protein ata concentration 5.4 mg/ml in 1:1 v/v ratio. Single crystals grewwithin 7 days to a typical a size of 0.15 � 0.15 � 0.05 mm.

A crystal was transferred into a cryosolution containing 60% 2-propanol, 0.1 M lithium sulfate, and 0.1 M sodium citrate pH 4.3.The crystal was harvested into a cryoloop in a 70% 2-propanolatmosphere and was cooled by transfer into liquid nitrogen. Datawas collected at 100 K on beamline I04 at the diamond light sourceto 2.65 Å overall resolution. The data was indexed and integratedin XDS11 and scaled using SCALA (CCP4).12,13 Chain A of the cas-pase-6 pH 4.5 model [PDB ID 2WDP] was used as search model

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Table 1Data processing and refinement statistics.

Parameter Z-VAD-FMK inhibited caspase-6

PDB ID 3QNWSpace group P21

Cell dimensions

a, b, c (Å) 87.21 65.45 91.72a,b,c (�) 90.0 91.24 90.0Resolution (Å) 29.1–2.65Rmerge

a (%) 11.4 (56.4)Mean I/rI a 14.8 (3.6)Completeness a (%) 99.2 (98.8)Multiplicity 7.5 (7.6)Refinement

No. reflections 28380Rwork/Rfree (%) 24.1/28.7B-factors

Protein (Å2) 29.7Ligand (Å2) 42.1Water (Å2) 21.5r.m.s.d.s

Bond lengths (Å) 0.005Bond angles (�) 0.815Ramachandran plot statistics

Favored (%) 96.4Allowed (%) 3.1Outlier (%) 0.5

a Values in parentheses are for highest resolution shell.

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for molecular replacement in PHASER (CCP4).14 The asymmetricunit of the caspase-6 Z-VAD-FMK complex contains four p10/p20subunits, reflecting two p102/p202 complexes. The molecularreplacement solution was subjected to one round of atomic refine-ment in REFMAC5 (CCP4).15 Initial electron density maps calcu-lated after the first round of refinement were inspected formissing protein residues. Residues of the loop L4 were not presentin the search model, but adopt a well-ordered conformation inchain A and C of the Z-VAD-FMK complex. After manual rebuildingof the L4 loop residues in Coot16 the model was subjected to an-other round of atomic refinement with isotropic B-factors. The

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electron density maps were further inspected for presence ofinhibitor peptide, and clear difference density was observed nearCys163 in chains A, C and G. Ligand structure and dictionary fileswere created in sketcher and the peptide was manually placed intothe electron density maps and real space refinement was per-formed in Coot. Water molecules were added with the water place-ment option in Coot. The structure geometry of the protein wasfinally checked using PROCHECK (CCP4) and Molprobity.17,18 Crys-tallographic data processing and refinement statistics have beensummarized in Table 1. Atomic coordinates and structure factorshave been deposited with the Protein Data Bank under the acces-sion code 3QNW.

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