doi:10.1016/j.jmb.2011.05.020 J. Mol. Biol. (2011) 410, 307–315

Contents lists available at www.sciencedirect.com

Journal of Molecular Biologyj ourna l homepage: ht tp : / /ees .e lsev ie r.com. jmb

A New Apo-Caspase-6 Crystal Form Reveals the ActiveConformation of the Apoenzyme

Ilka Müller1, Marieke B. A. C. Lamers1, Alison J. Ritchie1,Hyunsun Park2, Celia Dominguez2, Ignacio Munoz-Sanjuan2,Michel Maillard2 and Alex Kiselyov2⁎1BioFocus, Chesterford Research Park, Saffron Walden, Essex CB10 1XL, UK2CHDI Foundation, Inc., Suite 100, 6080 Center Drive, Los Angeles, CA 90045, USA

Received 4 February 2011;received in revised form12 May 2011;accepted 13 May 2011Available online20 May 2011

Edited by G. Schulz

Keywords:caspases;Huntington's disease;Alzheimer's disease;caspase-6;crystal structure

*Corresponding author. E-mail addrAlex.Kiselyov@CHDIFoundation.orAbbreviations used: HD, Hunting

mutant huntingtin; PDB, Protein Da

0022-2836/$ - see front matter © 2011 E

Caspase-6 has been identified as a key component in the pathway ofneurodegenerative diseases such as Alzheimer's disease and Huntington'sdisease. It has been the focus of drug development for some time, but onlyrecently have structural data become available. The first study identified anovel noncanonical conformation of apo-caspase-6 contrasting with thetypical caspase conformation. Then, the structures of both caspase-6zymogen and the Ac-VEID-CHO peptide inhibitor complex describedcaspase-6 in the canonical conformation, raising the question of why theintermediate between these two structures (mature apo-caspase-6) wouldadopt the noncanonical conformation. In this study, we present a newcrystal form of the apoenzyme in the canonical conformation by identifyingthe previous apostructure as a pH-inactivated form of caspase-6. Our newapostructure is further compared to the Ac-VEID-CHO caspase-6 inhibitorcomplex. The structural comparison allows us to visualize the organizationof loops L2, L3, and L4 upon ligand binding and how the catalytic grooveforms to accommodate the inhibitor.

© 2011 Elsevier Ltd. All rights reserved.

Introduction

Caspases or cysteinyl-aspartate-specific proteasesare critical components of apoptosis signalingpathways. Deregulation of their activity has beenassociated with a variety of conditions, includingcardiovascular, tumorigenic, and neurodegenera-tive disorders.1 It is generally accepted that extra-cellular apoptotic signals are transduced by twodistinct classes of caspases2,3—initiator and effectorcaspases4—that, as their names suggest, playdistinct roles in the cell death signaling relay.Initiator caspases include caspase-2, caspase-8,

ess:g.ton's disease; mHTT,ta Bank.

lsevier Ltd. All rights reserve

caspase-9, and caspase-10. Effector or ‘executioner’caspases are activated by initiator caspases and arerepresented by caspase-3, caspase-6, and caspase-7.Effector caspases cleave a multitude of functionalsubstrates, leading to organized cessation of cellularfunctions and ultimately cell death.5 The resultingproteolytic fragments themselves amplify the pro-apoptotic signal and thus need to be kept undertight control. In the context of the central nervoussystem, this leads to neuronal cell death, withaberrant effector caspase activity being associatedwith neurodegenerative diseases such as Alzhei-mer's disease and Huntington's disease (HD).6–10

Specifically for Alzheimer's disease, caspase-3 wasshown to process the amyloid precursor protein,leading to coprecipitation of cleaved fragments withamyloid plaques11 and excessive neuronal death.For HD, caspase-6 took central stage as a keyprocessing enzyme of mutant huntingtin (mHTT)protein, which features expanded polyglutamine

d.

308 X-ray Structure of Active Caspase-6

repeats at its N-terminus. mHTT cleavage atposition 586 reportedly produces “toxic” fragmentsthat contribute to HD pathology.12,13 This notion hasbeen further supported by in vivo evidence indicat-ing that administration of diverse neurotoxic xeno-biotics affected neither normal neuronal nor striatalfunctions in mice expressing caspase-6-resistant, butnot caspase-3-resistant, mHTT.14 It was concludedthat the specific blockage of caspase-6 proteolyticactivity may be neuroprotective in this diseasecontext.Significant attention has been paid to identify

potent and specific caspase inhibitors that featurefavorable pharmacokinetic and toxicology profilesthat consider specific therapeutic indication(s).Although numerous classes of caspase inhibitorshave been reported (for reviews, see Ivachtchenko etal.15 and Okun et al.16), their isoform specificityremains to be one of the main issues in the field.17,18Regardless of the specific chemical nature of theseinhibitors, they feature an electrophilic moiety (e.g.,carbonyl or fluoromethyl ketone group) that in-teracts with Cys163 at the active site, as well as asubstrate-mimetic recognition motif (e.g., tetrapep-tide, pentapeptide, or aryl pharmacophore). Themajority of the reported caspase-6 inhibitors arepeptide based. Unfortunately, these agents do notdisplay favorable pharmacokinetic profiles, namelystability and blood–brain barrier permeability.19

Isatin sulfonamide analogues that feature 2-fold to10-fold selectivity for caspase-6 over caspase-1,caspase-3, caspase-7, and caspase-8 isoforms havebeen described.20

Caspase-6 has been the focus of drug develop-ment (Chu et al.20 and references cited therein), butonly recently have structural data become available.The first structural study of apo-caspase-621 showedthe enzyme to be a constitutive p20/p10 dimer, withthe cleaved interdomain linker partially inserted inthe central groove of the p202/p102 tetramer.Distinct features of this apo-caspase-6 structureinclude a misaligned catalytic machinery and thelack of several structural elements required forsubstrate recognition. The same noncanonical con-formation has also been reported in a studycomparing the structures of apo-caspase-6 withand without the intersubunit linker.22 The firstreport on caspase-6 in the canonical caspaseconformation compared the caspase-6 zymogenwith the Ac-VEID-CHO-bound form,23 deducingthe mechanism of intramolecular self-cleavage.These structures raised the question of why matureapo-caspase-6 would adopt a noncanonical confor-mation, being the intermediate between caspase-6zymogen and the ligand-bound caspase-6, and inturn questioned whether this apostructure wasindeed physiologically relevant. To address thisquestion, we solved the structure of mature apo-caspase-6 in a new crystal form, which for the first

time shows the canonical caspase conformation alsofor apo-caspase-6. A further comparison with theAc-VEID-CHO caspase-6 inhibitor complex23 illus-trates the organization of loops L2, L3, and L4 uponligand binding and how the catalytic groove formsto accommodate the inhibitor.

Results and Discussion

It is generally recognized that effector caspasesundergo proteolytic cleavage of the inactivezymogen at a specific aspartate residue, resultingin a large N-terminal p20 polypeptide chain and asmall C-terminal p10 polypeptide chain, leading toa p202/p102 tetramer.24,25 In caspase-6, subunitsp20 and p10 comprise residues 24–179 and 194–293, respectively. The structure recently reportedfor the mature apo-caspase-6 also features a p202/p102 tetramer;21 however, it exhibits notabledifferences in the arrangement of several loopregions around the active site as compared to thecaspase-6 zymogen and the ligand-free crystalstructures of the closely related effector caspases,caspase-3 and caspase-7 (sequence identities of41% and 38%, respectively, and sequence similar-ities of 58% and 54%, respectively). In particular,residues forming a short anti-parallel β-sheet thatmediates substrate interactions in other caspasesare found to elongate the central α-helix, blockingthe canonical P1 substrate binding site anddissociating the His121/Cys163 catalytic histi-dine/cysteine dyad. Caspase-6 activity was con-firmed prior to crystallization of the target protein,and it is therefore presumed that the observedatypical misalignment of the active site could bean artifact of the crystallization procedure. Theprotein had been crystallized in the presence of0.1 M sodium acetate at pH 4.5. Caspase-6 activityis known to be optimal around neutral pH and isalmost entirely lost at pH 5 and below.26 Wespeculated that this pH dependence was reflectedin the reported crystal structure of caspase-6, andwe performed screens for crystallization conditionsnear the optimum pH for caspase-6 activity. Anew crystal form was obtained in the presence of0.1 M Tris (pH 7.4). The crystals exhibit themonoclinic space group P21 with four p202/p102tetramers in the asymmetric unit. The structurewas refined to a resolution of 2.53 Å with R-factorsof 20.8% and 26.8%, respectively (Table 1).

The overall structure of mature apo-caspase-6

Within the four caspase-6 p202/p102 tetramers inthe asymmetric unit, all of the eight p20/p10heterodimers are highly similar and can be super-imposed with RMSDs of between 0.26 and 0.36 Å for202 Cα atoms. For further discussion, we will refer to

Table 1. Data processing and refinement statistics

Parameter Apo-caspase-6

PDB ID 3P45Space group P21Cell dimensions

a, b, c (Å) 81.23, 161.24, 88.92α, β, γ (°) 90.0, 94.80, 90.0

Resolution (Å) 30–2.53Rmerge

a (%) 10.1 (55.0)Mean I/σIa 8.0 (2.0)Completenessa (%) 99.8 (99.8)Multiplicity 2.9

RefinementNumber of reflections 75,784Rwork/Rfree (%) 20.7/26.4B-factors

Protein 10.3Water 8.7

RMSDBond lengths (Å) 0.019Bond angles (°) 1.742a Values in parentheses are for the highest-resolution shell.

309X-ray Structure of Active Caspase-6

the tetramer consisting of chains A/B and C/D (Fig.1a). The overall topology of caspase-6 at pH 7.4closely resembles the caspase-6 zymogen structure[Protein Data Bank (PDB) ID: 3NR2; RMSD of 0.74 Åfor 205 Cα atoms within the p20/p10 dimer; Fig. 1b]and the mature ligand-free caspase-7 (PDB ID: 1K86;196 topologically equivalent Cα atoms within thep20/p10 dimer superimposing with an RMSD of0.85 Å; Fig. 1c), with a central six-stranded mixedβ-sheet flanked by five α-helices. It is distinct fromthe structure reported for caspase-6 at pH 4.6 (PDBID: 2WDP; RMSD of 3.3 Å for 203 Cα atoms withinthe p20/p10 dimer; Fig. 1d), leading to ourhypothesis that crystallization of caspase-6 at lowpH likely sequestered a pH-inactivated form, whichhad not been observed when caspase-3 or caspase-7was crystallized at pH b5 (i.e., PDB IDs: 2C1E and1KMC29). We will subsequently refer to the struc-tures obtained at pH 4.5 and pH 7.4 as low-pH andphysiological-pH mature apo-caspase-6 structures,respectively.The caspase-6 protein used for crystallization at

physiological pH comprised residues 24–179 and194–293 after self-cleavage of the expressed caspase-6zymogen. Caspase activity was confirmed prior tocrystallization (Fig. 2). The electron density is welldefined for residues Phe31-Cys163 in the p20subunit, residues Tyr198-Val212 and Thr222-Arg260, and residues Gln274-Lys291 in the p10subunit. Consequently, the first seven residues ofthe N-terminus of the p20 subunit, as well as the lasttwo residues of the p10 subunit, were absent fromthe electron density maps, and the loops constitut-ing the catalytic grooves L2 (residues 163–179), L3(212–222), and L4 (257–275) were found to bedisordered.

The L2′ loop

For inhibitor-bound caspase-3 and caspase-7, theconformation of the loops forming the catalyticgroove has been shown to be stabilized byinteractions with the cleaved interdomain linkerloop L2′ of the adjacent p20/p10 dimer;30,31

however, in the structure of the pro-caspase-7zymogen and the ligand-free caspase-7, the L2′loop is folded back and located at the interfacebetween the two p10 subunits of the p202/p102tetramer.32,33 In the apo-caspase-6 structure atphysiological pH, the L2′ loop is also orientedtowards the p10/p10′ interface (Fig. 3). It hasbeen proposed that the presence of inhibitor orsubstrate triggers flipping of the L2′ loop.34 Insupport of this hypothesis, apo-crystal structuresof caspase-3 and caspase-7 obtained in thepresence of inhibitor35,36 showed that the L2′loop folded against the L2 and L4 loops. This wasin contrast to the zymogen-like conformation ofthe L2′ loop in the previously reported apo-caspase-7 structure crystallized in the absence ofinhibitor.33 These studies on caspase-3 and cas-pase-7 suggest ligand-induced dynamics of theL2′ loop. Our crystallography-based insight intothe mature apo-caspase-6 and literature evidencefurther support the presence of multiple apostruc-tures that are likely to result from the inherentconformational freedom of the enzyme and couldbe stabilized by ligand binding. In the apo-caspase-6 crystals, the ligand binding site islocated at a large solvent channel through thecrystal and should be accessible to the ligand viasoaking. Furthermore, the conformation of the L4loop is not restricted by crystal packing; thus,rearrangement upon ligand binding should befeasible. Based on this insight, we soaked aninhibitor into the apo-caspase-6 crystals. Unfortu-nately, this approach failed; specifically, longersoaking times resulted in complete crystal decay,whereas shorter exposure to the molecule affordedlow compound occupancy. We propose thatwithin the crystal packing context, the L2′ loopin its locked position at the dimer interface isnot able to reorient towards, and to stabilize,the L4 loop in a conformation required forligand binding, as observed in the Ac-VEID-CHOcaspase-6 complex. (Further details can be foundin Supplementary Material.)

Comparison with the Ac-VEID-CHO caspase-6complex

In the Ac-VEID-CHO caspase-6 complex (PDB ID:3OD523), the L2′ loop is flipped by 180° and orientedtowards the L2/L4 loop interface of the neighboringp20/p10 dimer (Fig. 3). The conformation of loops L2and L3 closely resembles the one observed after the

Fig. 1. Overall structure of the mature apo-caspase-6 at physiological pH and in comparison with other apo-caspasestructures. For clarity, only one p20/p10 subunit is shown for the structure overlays. (a) Cartoon representation of thetetrameric assembly of the mature apo-caspase-6 at physiological pH, with the two p20/p10 dimers shown in blue andgreen, and red and orange, respectively. Loops composing the ligand binding site are largely disordered. (b) Overlay ofthe structure of the caspase-6 zymogen (PDB ID: 3NR2) with caspase-6 at physiological pH. In the zymogen structure, theuncleaved L2 loop (yellow) extends into the ligand binding site. (c) Overlay of the structures of mature apo-caspase-7(PDB ID: 1K86) and mature apo-caspase-6 at physiological pH. In this ligand-free caspase-7 structure, the L2 loop(highlighted in yellow for caspase-7) is located at the p10/p10′ interface. (d) Overlay of apo-caspase-6 at pH 4.6 (PDB ID:2WDP) with mature apo-caspase-6 at physiological pH (gray). Conformational rearrangement at low pH occurs near theactive groove, with residues 61–67 and 126–138 adopting a helical conformation (highlighted in cyan). The figures werecreated in PyMOL27 and CCP4mg.28

310 X-ray Structure of Active Caspase-6

ligand soaking of the apo-caspase-6 crystals, asdescribed above. The L4 conformation is stabilizedvia interactions with loops L2 and L2′ at the base; itscrown extends away from the core of the molecule

and is separated from the adjacent tetrapeptidemolecule by a layer of water molecules, thereforeshowing its accessibility from solvent space (Fig. 4).Interestingly, Cys264, which is part of the L4 loop, is

Fig. 2. Caspase-6 activity data for the inhibitory peptideAc-VEID-CHO.

311X-ray Structure of Active Caspase-6

one of four surface cysteine residues for whichcacodylation has been observed in the crystalstructure of the Ac-VEID-CHO caspase-6 complex.The complex had been crystallized in the presence ofDTT and sodium cacodylate, amixture that had been

previously reported to lead to the formation of theS-(dimethylarsenic) cysteine adduct.37 Since theprotein was incubated with the inhibitor prior tocrystallization, it is reasonable to assume that the Ac-VEID-CHO caspase-6 complex was formed prior tocacodylate exposure. This is in agreement with theobservation that the active site Cys163, whichappears to be accessible to covalent modification inthe apocomplex but is blocked after peptide binding,is not cacodylated. The residue has to be accessible toallow for reactivity at Cys264; therefore, it seemsunlikely that the L4 loop is packed tightly against thepeptide in the cacodylate-free complex. Based on thisobservation, we believe that solvent separationbetween the peptide and the L4 loop is a genuinefeature of the Ac-VEID-CHO caspase-6 complex thatdistinguishes caspase-6 from caspase-3 and caspase-7.In the Ac-DEVD-CHO caspase-3 complex (PDB ID:2H5I38), a hydrogen bond between the L4 loop andthe P4 side chain is observed. In the Ac-DEVD-CHOcaspase-7 complex (PDB ID: 1F1J31), we observedseveral direct hydrogen bonds to the L4 loopinvolving the P4 side chain and the backbone(Fig. 4). We also believe that the reported structureof the Ac-VEID-CHO caspase-6 complex is relevantto a physiologically active enzyme. In view of thewell-defined network of intermolecular interactionsdescribed in this work,23 we consider it immediatelysuitable for a structure-based drug discovery ofpotent and selective caspase-6 inhibitors.

Fig. 3. Comparison of the overallstructure of mature apo-caspase-6at physiological pH with that ofcaspase-6 with bound Ac-VEID-CHO peptide (PDB ID: 3OD5).Cartoon representation of caspase-6in complex with Ac-VEID-CHO(light gray). Flexible surface loopsare highlighted. The apo-caspase-6structure at physiological pH isoverlaid in dark gray. The figurewas created in PyMOL.27

Fig. 4. Comparison of inhibitor-bound caspase-3, caspase-6, and caspase-7 structures. Interaction of the L4 loopresidues with the P4 site of bound peptide in the Ac-VEID-CHO caspase-6 (PDB ID: 3OD5; gray), Ac-DEVD-CHOcaspase-3 (PDB ID: 2H5I; magenta), and Ac-DEVD-CHO caspase-7 (PDB ID: 1F1J; blue) complexes, respectively. Incontrast to the caspase-3 and caspase-7 complexes, all hydrogen bonds between the L4 loop and the inhibitor peptide arewater mediated in the caspase-6 complex. The figures were created in PyMOL.27

312 X-ray Structure of Active Caspase-6

In conclusion, this article describes the structuresof caspase-6 in its apo form at physiological pH. Itclosely resembles mature ligand-free caspase-7 witha central six-stranded mixed β-sheet flanked by fiveα-helices. Loops L2, L3, and L4, which constitute theligand binding site in the Ac-VEID-CHO caspase-6complex, are disordered, and the L2′ loop resides atthe p10/p10′ interface as observed in the caspase-6zymogen structure. Our results indicate that thepresence of ligand induces loop L2′ to reorienttowards, and to stabilize, the L2 and L4 loops of theneighboring p20/p10 dimer, with the L2′ fliprequired for ligand binding. The overall topologyof the apostructure at physiological pH is distinctfrom the structural data previously reported forcaspase-6 at pH 4.5. Even though its noncanonicalfold does not impair inhibitor binding, we think thatit does not represent a physiologically relevantconformation that would provide an allosteric sitefor drug development.

Materials and Methods

Gene construction and protein expression

Oligonucleotides were designed to amplify the cata-lytic domain (amino acid residues 24–293) of humancaspase-6 for cloning into a BioFocus in-house expres-sion vector. cDNA products of the correct size forcaspase-6 were obtained from polymerase chain reactionexperiments using cDNA encoding full-length caspase-6.Products were successfully cloned into the BioFocusvector pT7-CH for Escherichia coli expression under thecontrol of the T7 promoter. The vector–insert combina-tion provided a C-terminal hexa-histidine purificationtag on the expressed protein. For large-scale expression,recombinant human caspase-6 was expressed in RosettaE. coli cells grown overnight at 37 °C from glycerol

stock. The next day, 6×1 L cultures were inoculatedwith 50 ml of overnight starter cultures. Cells weregrown at 37 °C until an OD600 of 1.2 had been reached.Cells were cooled to 25 °C, induced with 0.5 mM IPTG,and shaken overnight before being harvested. Cellpellets were harvested, washed with phosphate-bufferedsaline, and then stored at −80 °C. We found that thetemperature had clearly an effect on the yield of fullyprocessed caspase-6 protein, as induction at 25 °C,compared to 37 °C, resulted in higher yields. In addition,increased protein yields were also obtained when thecells were induced at a higher cell density.

Protein purification

Cell paste from 6×1 L cultures was resuspended on icein 100 ml of 25 mM Tris (pH 8.0) containing 25 mMimidazole, 100 mM NaCl, 10% glycerol, and 0.1% 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonicacid. Following mechanical disruption of the cells, thesoluble fraction was harvested by centrifugation at60,000g for 30 min. The cleared supernatant was incubatedbatchwise with 0.5 ml of NiNTA (Qiagen) for 2 h at 4 °C toallow binding. The resin was collected by centrifugation at400g and washed once with buffer A [25 mM Tris (pH 8.0)containing 25 mM imidazole, 100 mMNaCl, 10% glycerol,and 0.1% 3-[(3-cholamidopropyl)dimethylammonio]pro-panesulfonic acid] before being loaded into an Omnifitcolumn. The column was attached to an ÄKTAexpress,run through IMAC elution (using buffer A supplementedwith 500 mM imidazole and 10 mM DTT), and thensubjected to size-exclusion chromatography [with aSuperdex 200 16/60 column equilibrated in 20 mMsodium acetate (pH 5.5) containing 50 mM NaCl and10 mM DTT], as the protein was most stable for storageunder these conditions. After size-exclusion chromatog-raphy, the fractions were collected and analyzed by SDS-PAGE stained with Coomassie brilliant blue. The purestfractions were pooled, concentrated to 8.2 mg/ml byultrafiltration, and used for crystallization. DTT (10 mM)was added to the concentrated protein prior to crystalli-zation. Protein concentration was determined with

313X-ray Structure of Active Caspase-6

Coomassie Plus reagent, measuring optical absorbance at595 nm.

Protein activity

Caspase-6 activity was confirmed using an assay that isbased on the protolytic cleavage of a fluorogenic substrate.This substrate consists of two caspase-6 recognitionpeptide molecules that are covalently linked to twoamine groups of the fluorescent dye rhodamine 110(Z-VEID-R110; Invitrogen), which suppresses R110 fluo-rescence. During proteolysis, both recognition peptidesare cleaved off, and subsequent dequenching of the dyeindicates enzymatic activity. Pipes (20 mM; pH 7.4),100 mM NaCl, 0.03% Pluronic®, 10% sucrose, 1 mMethylenediaminetetraacetic acid, and 5 mM glutathionewere used as assay buffer. The enzyme concentration wasdetermined with Coomassie Plus reagent, measuringoptical absorbance at 595 nm. Recombinant caspase-6activity was detected by measuring the R110 release fromZ-VEID-R110 at 37 °C using a Perkin-Elmer EnVision® atan excitation wavelength of 485±14 nm and at anemission wavelength of 535±25 nm. The enzyme prepa-ration used in the enzymatic studies was titrated using thesubstrate Z-VEID-R110 andwas found to be active (resultsnot shown). The inhibitor concentration that results in 50%inhibition (IC50) was determined for the reported caspase-6-competitive and caspase-6-reversible inhibitor Ac-VEID-CHO.39,40 The inhibitor was resuspended in dimethylsulfoxide, serially diluted in assay buffer, and combinedwith 1 pg of caspase-6 on a 96-well plate. The maximumdimethyl sulfoxide concentration was 1%, and thecaspase-6 substrate was used at a concentration of 10 μM.

Crystallization and data collection

Crystals of mature apo-caspase-6 were obtained withhanging-drop vapor diffusion on 24-well plates (VDXm;Hampton Research) at 20 °C by mixing 1.0 μl of proteinsolution [in 20 mM sodium acetate (pH 5.5), 50 mM NaCl,and 0.5 mM Tris(hydroxypropyl)phosphine] with 1.0 μl ofreservoir solution (0.5 ml) consisting of 3.3 M sodiumnitrate, 0.1 M Tris (pH 7.4), 0.5% ethyl acetate, and 5 mMTris(hydroxypropyl)phosphine. Single crystals(0.5 mm×0.3 mm×0.1 mm) grew by microseedingstraight into the drop after setup within 1 week at 20 °C.The crystallization drop was overlaid with 3 μl ofcryoprotectant containing 20% ethylene glycol, 3.5 Msodium nitrate, and 0.1 M Tris–HCl (pH 7.4), and a crystalwas harvested for data collection. The crystal was flashfrozen in liquid nitrogen, and X-ray data collection wascarried out at 100 K on a Rigaku R-Axis IV image platedetector, with data indexed, integrated, and scaled usingMOSFLM and SCALA (CCP4),41–43 respectively.

Structure solution and refinement

Chain A of the pH-inactivated caspase-6 model com-prising residues 31–293, representing one p20/p10 dimer,was used for molecular replacement using Phaser,44

locating eight p20/p10 subunits in the asymmetric unit.The resulting model was given two rounds of atomic

refinement with tight geometric weights usingREFMAC5.45 The electron density maps calculated aftermolecular replacement and initial refinement were exam-ined, and residues with a poor fit to the electron densitymap were omitted from the model. The truncated modelwas used as a starting model for automated modelbuilding in Buccaneer.46 Possibly due to the disorder ofa large number of surface loops and hence chain breaksaround the active site, automated model building failed toimprove the initial model. In an alternative approach,NCS-averaged electron density maps were calculated inPARROT47 and used to rebuild missing residues manuallyin Coot.48 The resulting extended model was then refinedusing REFMAC5. To account for differences in thermalmotion, we performed TLS refinement on the completedmodel, with one TLS group per p10/p20 heterodimer.Water molecules were then added using the waterplacement option in Coot and refined using REFMAC5.For all eight heterodimer molecules in the asymmetricunit, chain breaks are observed between ~Ala162 andTyr198, between ~Val212 and Thr222, and between~Arg260 and Gln274, and the residues were omittedfrom the model. Structural geometry was checked usingPROCHECK and MolProbity.49,50

Accession number

Atomic coordinates and structure factors have beendeposited in the PDB under accession code 3P45.

Supplementary Data

Supplementary data associated with this articlecan be found, in the online version, at doi:10.1016/j.jmb.2011.05.020

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