The molecular basis of ubiquitin-like protein NEDD8deamidation by the bacterial effector protein CifAllister Crowa, Richard K. Hughesa, Frédéric Taiebb,c,d,e, Eric Oswaldc,d,e,f,g, and Mark J. Banfielda,1

aDepartment of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, United Kingdom; bINP-ENV de Toulouse, F-31076Toulouse, France; cInstitut National de la Recherche Agronomique, USC 1043, F-31300 Toulouse, France; dInstitut National de la Santé et de la RechercheMédicale, Unité Mixte de Recherche 1043, F-31300 Toulouse, France; eCentre de Physiopathologie de Toulouse Purpan, Université Paul Sabatier de Toulouse,F-31400 Toulouse, France; fCentre National de la Recherche Scientifique, Unité Mixte de Recherche 5282, F-31400 Toulouse, France; and gService deBactériologie-Hygiène, Hôpital Purpan, Centre Hospitalier Universitaire de Toulouse, F-31059 Toulouse, France

Edited by Scott J. Hultgren, Washington University School of Medicine, St. Louis, MO, and approved May 15, 2012 (received for review July 25, 2011)

The cycle inhibiting factors (Cifs) are a family of translocatedeffector proteins, found in diverse pathogenic bacteria, thatinterfere with the host cell cycle by catalyzing the deamidationof a specific glutamine residue (Gln40) in NEDD8 and the relatedprotein ubiquitin. This modification prevents recycling of neddylatedcullin-RING ligases, leading to stabilization of various cullin-RING li-gase targets, and also prevents polyubiquitin chain formation. Here,we report the crystal structures of two Cif/NEDD8 complexes, re-vealing a conserved molecular interface that defines enzyme/sub-strate recognition. Mutation of residues forming the interfacesuggests that shape complementarity, rather than specific individ-ual interactions, is a critical feature for complex formation. Weshow that Cifs from diverse bacteria bind NEDD8 in vitro and con-clude that they will all interact with their substrates in the sameway. The “occluding loop” in Cif gates access to Gln40 by forcinga conformational change in the C terminus of NEDD8. We usednative PAGE to follow the activity of Cif from the human pathogenYersinia pseudotuberculosis and selected variants, and the posi-tion of Gln40 in the active site has allowed us to propose a catalyticmechanism for these enzymes.

bacterial pathogenesis | cyclomodulins | host cell manipulation | structuralbiology | type III secreted effector proteins

Many pathogenic Gram-negative bacteria use a type IIIsecretion (T3S) system to translocate effector proteins

into target cells (1). Once inside the host cell, effectors act tosubvert and/or otherwise manipulate vital cellular systems andrepresent a key virulence strategy for these pathogens (2). TypeIII secreted effectors (T3SEs) can encode a wide range of dif-ferent enzymatic activities (3). Because of the generally lowamino acid sequence conservation of effectors to proteins ofknown function, these activities are often only identifiedthrough structural studies.In the past decade, progression of the host cell cycle has

emerged as one cellular system targeted by multiple T3SEs fromdiverse pathogens (4–6). The cycle inhibiting factors (Cifs)comprise a family of T3SEs from animal pathogens and insectsymbionts (7) that induce a cytopathic phenotype in host cellsthat includes cell-cycle arrest at the G2/M or G1/S transition (6,8–12). It has been suggested that during the infection process,restriction of the host cell cycle might delay epithelial cell re-newal and favor gut colonization (7). Recently, regulation ofubiquitin-mediated proteolysis has been implicated in themechanism of Cif-induced cell-cycle arrest (7). Analysis of hostcell proteins regulating cell-cycle checkpoints revealed accumu-lation of cyclin-dependent kinase inhibitors p21Waf1/Cip1 andp27Kip2 in response to Cifs (10, 11); these proteins are usuallydegraded by ubiquitin-mediated proteolysis. Further, ubiquitin-mediated proteolysis of GFP reporters expressed in HeLa cellswas blocked following delivery of Cif from Burkholderia pseu-domallei (CifBp) (9).One mechanism for managing eukaryotic cell-cycle pro-

gression is timed degradation of key regulators through ubiq-

uitinylation and targeting to the 26S proteasome (13). In thissystem, ubiquitin molecules are covalently attached to proteinsdestined for destruction by the concerted action of an E1-E2-E3enzyme cascade, with substrate specificity defined by E3 ligases(14). The largest family of E3 ligases is the cullin RING E3ubiquitin ligases (CRLs) (15). As befitting their critical role inmany cellular processes, the activities of CRLs are tightly regu-lated. One mechanism for CRL activation is through conjugationof the ubiquitin-like molecule NEDD8 (neural precursor cellexpressed, developmentally down-regulated 8) to the cullinsubunit (neddylation) (16, 17), stimulating substrate ubiquitina-tion. Importantly, cycling of CRLs between neddylated anddeneddylated forms is required for full activity (18, 19).Cif from enteropathogenic Escherichia coli (CifEc) interacts

with NEDD8 in both yeast two-hybrid assays (20) and Proto-Array analysis (21). CifEc also colocalizes with NEDD8 in thenuclei of HeLa cells (20) and specifically binds to neddylatedCRLs, but not to the unmodified proteins, in immunoprecipita-tion assays (20). Significantly, CifEc was shown to inhibit the E3ligase activity of neddylated cullins (7, 9, 20, 21). Cif activity iscorrelated with accumulation of CRLs in their neddylated forms(22), preventing the neddylation/deneddylation cycle and lockingCRLs in a neddylated but inactive form. This leads to stabiliza-tion of numerous CRL targets in cells, which presumably triggersthe downstream cytopathic phenotype.Structural studies of CifEc, CifBp, and Cif from Photorhabdus

luminescens (CifPl) (12, 23, 24) revealed a common fold, despitesharing low overall sequence identity. The proteins comprisea head-and-tail domain structure reminiscent of a comma orapostrophe. The C-terminal head domain comprises a cysteineprotease-like fold and contains a conserved Cys-His-Gln catalytictriad. Regions of the N-terminal tail domain are important for Ciffunction, and it has been hypothesized that they contribute tosubstrate recognition (12, 20). A fundamental advance in un-derstanding the mechanism by which Cifs inhibit CRL activityemerged when these effectors were shown to possess a specificdeamidase activity (9). CifBp and CifEc both catalyze the deami-dation of Gln40 in NEDD8 (converting this residue to a Glu).CifBp also efficiently deamidates Gln40 of ubiquitin. Ectopic ex-pression of a NEDD8(Gln40Glu) mutant in HeLa cells led tostabilization of CRL substrates and generated an equivalent effect

Author contributions: A.C. and M.J.B. designed research; A.C., R.K.H., and F.T. performedresearch; A.C., R.K.H., F.T., E.O., and M.J.B. analyzed data; and A.C. and M.J.B. wrotethe paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: Atomic coordinates and structure factors reported in this paper havebeen deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 4F8C–4FBJ).1To whom correspondence should be addressed. E-mail: mark.banfield@jic.ac.uk.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1112107109/-/DCSupplemental.

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to that exhibited by Enteropathogenic E. coli (EPEC) infection(9). This provides strong evidence that Cif deamidase activity to-ward NEDD8-Gln40 is necessary and sufficient for the Cif-me-diated cytopathic phenotype.The purpose of this study was to investigate the interaction

between Cifs and NEDD8, both biochemically and structurally,and also to probe the mechanism of deamidation. Here, wereport the crystal structures of two Cif/NEDD8 complexes, oneincluding Cif from the human pathogen Yersinia pseudotuber-culosis (CifYp) and the second from the insect symbiontP. luminescens. The structure of CifYp has not been determinedbefore. The two complexes share a common mode of bindingwith interactions arising from both the head and tail domains.The Gln40 substrate residue of NEDD8 extends into the cata-lytic site of the Cifs. We also show that other members of the Ciffamily bind to NEDD8 and suggest that our structures area model for all Cif/NEDD8 complexes. Using site-directedmutagenesis of CifYp, we have probed the enzyme/substrate-binding interface and hypothesize that overall shape comple-mentarity rather than any specific individual interaction is thedriving force behind complex formation. Finally, we have alsoinvestigated the catalysis of NEDD8 and ubiquitin deamidationby CifYp. This work has allowed us to propose a mechanism forthe activity of Cifs that ultimately leads to inhibition of the hostubiquitin-dependent proteasomal degradation pathway and thecytopathic phenotype.

ResultsCifs Crystallize in a 1:1 Complex with NEDD8. To define how the Ciffamily of effectors (sequence alignment is shown in Fig. 1)engages their substrate and catalyzes the deamidation ofNEDD8, we attempted to crystallize a variety of Cif/NEDD8complexes (details of gene cloning and protein expression areprovided in Materials and Methods). We obtained diffraction-quality crystals of catalytic site mutants CifPl(Cys123Ser) andCifYp(Cys117Ala) in complex with NEDD8 following copur-ification of the enzyme and substrate (Materials and Methods,Fig. 2, Fig. S1, and Table S1). Both structures were solved bymolecular replacement using uncomplexed Cifs and NEDD8 assearch models. X-ray data collection, refinement, and validationstatistics are given in Table 1. The CifPl(Cys123Ser)/NEDD8 andCifYp(Cys117Ala)/NEDD8 crystal structures comprise one andtwo 1:1 complexes of Cif and NEDD8 in the asymmetrical unit,respectively. Each of these complexes shows very similar overallarrangements with rmsds (25) of 0.59 Å between the two com-

plexes in the CifYp(Cys117Ala)/NEDD8 crystal and 1.41/1.47 Åbetween the CifPl(Cys123Ser)/NEDD8 and the two CifYp(Cys117Ala)/NEDD8 complexes (320, 279, and 284 equivalentCα atoms considered). These structures most likely representa substrate-binding mode that is conserved across the Cif family.A cartoon representation of the CifYp(Cys117Ala)/NEDD8complex (henceforth CifYp/NEDD8) is shown in Fig. 3A.

Crystal Structure of CifYp. The crystal structure of free CifYp hasnot been reported previously. Although in complex withNEDD8, the structure of CifYp is very similar to other Cifs. Itoverlays on CifBp (23), CifPl (23), and the truncated structure ofCifEc (24) with rmsds (25) of 1.69 Å, 1.72 Å, and 1.20 Å (231,229, and 167 equivalent Cα atoms considered). The catalytic triadresidues Cys117, His173, and Gln193 (CifYp numbering) occupyessentially equivalent positions in all structures.

An Extensive Binding Interface Is Formed Between CifYp/Pl and NEDD8.Structures of uncomplexed Cifs have been described as compris-ing a head-and-tail domain structure reminiscent of a commaor apostrophe (23). The structures of CifYp/NEDD8 and CifPl(Cys123Ser)/NEDD8 independently show that both the headdomain [which comprises residues Val116/122 (CifYp/CifPl) to theC terminus] and tail domain [which comprises residues from theN terminus to Pro115/121 (CifYp/CifPl)] make significant con-tributions to the NEDD8 binding interface (Fig. 3 A–D). Thisinterface buries 1508.5 Å2 of the NEDD8 solvent-accessible sur-face area in the CifYp/NEDD8 complex, equivalent to 31.1%of the total [1,376.1 Å2 (29.2% of the total) is buried in theCifPl(Cys123Ser)/NEDD8 complex]. Both the CifYp/NEDD8 andCifPl(Cys123Ser)/NEDD8 interfaces have a complexation signifi-cance score of 1.00 as defined by Protein Interfaces, Surfaces andAssemblies (PISA) (26). NEDD8 residues that form the interfacewith Cif reside on the loop between β1 and β2 (Lys6–Lys11), α1and β3 (Glu31–Arg42, which contains the substrate Gln40 resi-due), β3 and β4 (Ile44–Gly47), and the C-terminal β-strand(Val66–Arg74). These four regions form interactions with the tail,head, tail, and tail-and-head domains of Cif, respectively. Fulldetails of the residues contributing to the interfaces and theinteractions they form are given in Tables S2–S5.

Changes in Conformation on Complex Formation. The availability ofthe uncomplexed CifPl (23) and NEDD8 (27) structures, along-side the CifPl(Cys123Ser)/NEDD8 complex (henceforth CifPl/NEDD8) described here has allowed analysis of the conforma-

Fig. 1. Numbered sequence alignment, including four members of the Cif family. Highlighted in blue are the residues of the catalytic triad. Highlighted inred (tail domain) and green (head domain) are residues in CifYp that were mutated to investigate either NEDD8 binding and/or catalysis. The residue that maybe involved in NEDD8/ubiquitin selectivity is shown in yellow. Asterisks denote positions of identical residues.

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tional changes occurring during complex formation. Overall, thestructures of CifPl and NEDD8 are very similar in their unboundand bound forms (rmsds of 0.86 Å and 0.62 Å, using 247 and 71equivalent Cα atoms, respectively) (25) (Fig. 4). The only notablechange in CifPl on binding NEDD8 is a slight reorientation at thetip of the tail domain (residues Ile94–Tyr116) that bends towardthe substrate protein (maximum Cα shift of 3.4 Å for Glu100). InNEDD8, the C-terminal five residues (Val70–Arg74) undergoa significant shift on binding CifPl (9.7 Å for the Cα of Arg74)that breaks β-sheet hydrogen-bonding interactions and displacesthe C-terminal residues away from the α1/β3 loop (Fig. 4). Theα1/β3 loop contains the substrate NEDD8:Gln40 residue, and thedisplacement of the C terminus appears critical for positioningthis residue in the Cif active site (Fig. 3 D–F). CifPl residuesVal122 and Leu203 are the key residues that enable the dis-placement of this region. The same displacement of the C ter-minus of NEDD8 is observed in the CifYp/NEDD8 complex withVal116 and Leu196 substituting for Val122/Leu203 (Fig. 3 D–F).This Val/Leu pair is conserved in all Cifs except CifEc, where theequivalent Val is a Ser residue.

Cif Proteins Bind NEDD8 in Solution.Using gel filtration, we showedthat CifYp, CifEc, CifBp, and CifPl (all containing mutations in theactive site Cys) were able to bind NEDD8, with the increases inapparent molecular mass on complex formation consistent with1:1 binding (Fig. 2, Fig. S1, and Table S1). We then focused onthe CifYp/NEDD8 interaction as a model system to probe theproperties of Cif/NEDD8 binding attributable to the availabilityof the crystal structure and this pathogen’s relevance forhuman disease. We quantified the interaction between CifYp(Cys117Ala) and NEDD8 using isothermal titration calorimetry(ITC). This experiment showed the affinity was in the sub-micromolar range (Fig. 5 and Table S6). The ITC data in-

dependently confirmed the 1:1 binding stoichiometry previouslyobserved by gel filtration, further supporting the validity of theinteraction observed in the crystals. The interaction betweenCifYp(Cys117Ala) and NEDD8 was also analyzed using intrinsictryptophan fluorescence (SI Text and Figs. S2 and S3).

Targeted Mutagenesis of the Cif/NEDD8 Binding Interface Disruptsthe Interaction. Having quantified the interaction betweenCifYp(Cys117Ala) and NEDD8, we investigated the importanceof interfacing residues identified in the CifYp/NEDD8 struc-ture. First, we constructed five independent alanine sub-stitution mutants in residues that we hypothesized would formimportant interactions with NEDD8 (CifYp tail domain resi-dues Asp66 and Asp67 and CifYp head domain residuesAsn122, Asn167, and Leu196; Fig. 3 B and C and Tables S2–S5). All but CifYp(Asn122Ala) were expressed and purified, anddisplayed comparable chemical unfolding/refolding profiles toWT protein; therefore, these mutations do not significantlyaffect stability (Fig. S4 and Table S7). Somewhat surprisingly,each of these mutants, which included examples of removingintermolecular hydrogen bonds and hydrophobic interactions,gave only marginal decreases in the affinities as measured byITC (Fig. 5B and Table S6); similar results were obtained usingfluorescence-based assays (Fig. S2 and Table S6).We then made six additional mutants in the CifYp(Cys117Ala)

background to test the effects on complex formation of in-troducing steric clashes and/or charged residues at interfacepositions. Two of these mutations [Asp67Arg and Val104Glu(tail domain)] were designed as controls and targeted residuesthat did not closely associate with NEDD8 in the structure, andfour [Asp66Arg and Leu106Glu (tail domain) and Val116Aspand Gly118Thr (head domain)] were predicted to compromiseCifYp/NEDD8 interaction (Fig. 3C). All variant proteins exceptAsp67Arg were expressed and purified, and displayed comparablechemical unfolding/refolding profiles to WT protein (Table S7).Although the affinity of the interaction between CifYp(Val104Glu)and NEDD8 was equivalent to WT, CifYp(Leu106Glu), CifYp(Val116Asp), and CifYp(Gly118Thr) were each severely compro-mised in NEDD8 binding as measured by ITC (Fig. 5B andTable S6); similar results were obtained using fluorescence(Fig. S2 and Table S6). CifYp(Asp66Arg) showed an intermediateeffect but was still significantly impaired in binding to NEDD8.

NEDD8 Residue Glutamine 40 Occupies the Cif Active Site. Thestructures of CifYp/NEDD8 and CifPl/NEDD8 revealed that thesubstrate NEDD8 residue Gln40 projects from the loop betweenα1 and β3 into the Cif active site (Fig. 3F). In the CifPl/NEDD8complex, the Nε2 atom of NEDD8:Gln40 forms a hydrogen bondto the OH group of Ser123 (the mutated catalytic residue); it isunlikely that this represents a catalytically competent orientationfor this side chain. In the CifYp/NEDD8 complex, the Cys117Alamutation allows the side chain of NEDD8:Gln40 to lie directlyover what would be the catalytic center. We produced a model ofthe WT CifYp/NEDD8 complex by mutating (in silico) Ala117back to a Cys (Fig. 3F). In this model, the thiol group of Cys117is well-positioned for nucleophilic attack of the NEDD8:Gln40Cδ atom, which would initiate the deamidation reaction.From the structures of the complexes, we also identified ad-

ditional Cif residues that may be relevant for the catalytic ac-tivity. Foremost among these is Asp195 (CifYp numbering), aresidue conserved in all Cifs. The Asp195-Oδ1 atom forms a hy-drogen bond with NEDD8:Gln40Nε2 (2.81 Å) in the CifYp/NEDD8 complex (Fig. 3F). Other interactions in the CifYp/NEDD8 active site include hydrogen bonds between NEDD8:Gln40Oδ1 with the backbone amides of Cys117 (2.77 Å) andLeu196 (2.94 Å) and NEDD8:Gln40Nε2 with the backbone car-bonyl of Gly172 (3.13 Å). The roles of these residues in orientingthe NEDD8(Gln40) side chain are discussed below.

Fig. 2. Gel filtration enables purification of a 1:1 complex of CifYp andNEDD8. (A) Gel filtration traces derived from purifications of CifYp, NEDD8,and the complex. Details of the elution volumes are given in Table S1. mAU,milli-absorbance units. (B) SDS/PAGE analysis of fractions collected across theelution peaks corresponding to the CifYp/NEDD8 complex and excess NEDD8(from the same gel filtration experiment).

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NEDD8 and Ubiquitin Deamidation by CifYp. Using tryptic digestliquid chromatography MS, we confirmed that the only modifi-cation to NEDD8 from exposure to CifYp was the conversion ofGln40 to a Glu. We then used native PAGE (nPAGE) to in-vestigate the deamidase activity of CifYp (9, 28). This assay ena-bles screening of selected structure-informed mutants for effectson activity. Incubation of NEDD8 with CifYp results in a shift inelectrophoretic mobility equivalent to NEDD8(Gln40Glu); noshift occurs for NEDD8(Gln40Ala) (Fig. 6A). Further, this shiftis dependent on the catalytic residue Cys117 [incubation withCifYp(Cys117Ala) does not result in a shift] (Fig. 6A).We also tested the activity of the CifYp(Leu106Glu) and

CifYp(Asp195Asn) variants using the nPAGE assay. TheCifYp(Leu106Glu) mutation compromises the interaction ofCifYp(Cys117Ala) with NEDD8, despite its position near thetip of the tail domain, distant from the active site. Consistentwith this, catalytic activity for the CifYp(Leu106Glu) variant issignificantly reduced compared with WT (Fig. 6A). The struc-tures of CifYp/NEDD8 and CifPl/NEDD8 suggested a putativerole for Asp195 (CifYp numbering) in catalysis. We found thatthis variant still retained a significant level of activity, suggestingthat an Asp at this position is not critical for function and isunlikely to act as a general acid/base in catalysis (Fig. 6A).We also tested the ability of CifYp, CifYp(Cys117Ala), and

CifYp(Leu106Glu) to deamidate ubiquitin (Fig. 6B). Similar toNEDD8, we observed an electrophoretic shift of ubiquitin in thepresence of the WT enzyme but not with CifYp(Cys117Ala)or CifYp(Leu106Glu).To obtain further details of CifYp’s deamidase activity, we

performed enzyme titration experiments (9, 28) with NEDD8

and ubiquitin (Fig. 6C and Fig. S5). These assays show thatNEDD8 is a better substrate for CifYp than ubiquitin (∼0.025pmol of CifYp required for complete conversion of 350 pmol ofNEDD8 in the assay compared with ∼0.25 pmol needed forubiquitin). This is consistent with previous studies of CifBp andCifEc that also show a preference for NEDD8 (9). To supportthese results, we also performed enzyme titration experimentswith NEDD8 and the CifYp(Cys117Ala), CifYp(Leu106Glu), andCifYp(Asp195Asn) variants (Fig. 6C and Fig. S5).

p21 Accumulates in HeLa Cell Culture in the Presence CifYp. Deliveryof purified Cif proteins to HeLa cells (CifEc, CifBp, and Cif fromPhotorhabdus species) results in the accumulation of cell-cycleregulators, including p21 and p27, with this activity dependent onthe active site cysteine (10). We have shown that WT CifYp alsostabilizes p21, although the effect was weaker than that observedfor CifEc (Fig. 7). Both CifYp(Cys117Ala), a catalytic site muta-tion, and CifYp(Leu106Glu), a tail domain mutation distant fromthe active site (that severely compromises CifYp/NEDD8 in-teraction and catalysis in vitro), also prevent p21 accumulation inHeLa cells (Fig. 7).

DiscussionPathogenic bacteria of both animals and plants have evolvedmechanisms for directly modifying host cell targets through thedelivery of enzymes by the T3S system. These enzymes encodea variety of activities (3). Hydrolytic activity is emerging as a keymechanism used by T3SEs, with many examples of proteases(29–33) and phosphatases (34, 35) acting in host cells.

Table 1. X-ray data collection, refinement, and validation statistics

CifPl/NEDD8 (in-house) CifPl/NEDD8 (DLS-I02) CifYp/NEDD8 (DLS-I24)

Data collectionSpace group P21 P21 P6322Cell dimensions

a, b, c; Å 40.7, 56.0, 67.6 40.7, 56.1, 67.6 125.4, 125.4, 169.9α, β, γ; ° 90.0, 104.0, 90.0 90.0, 104.0, 90.0 90.0, 90.0, 120.0

Resolution, Å 19.10–2.10 (2.21–2.10) 42.60–1.60 (1.69–1.60) 66.90–1.95 (2.06–1.95)Rmerge 0.039 (0.123) 0.052 (0.288) 0.079 (0.463)I/σ(I) 27.7 (12.7) 11.5 (3.0) 18.5 (4.2)

Completeness, % 95.0 (93.3) 96.4 (96.2) 100.0 (100.0)Multiplicity 6.1 (5.9) 4.4 (4.3) 11.1 (9.2)

RefinementResolution, Å — 38.20–1.60 (1.64–1.60) 66.90–1.95 (2.00–1.95)

Rwork, % — 16.5 (26.3) 18.5 (22.1)Rfree, % — 23.6 (31.5) 23.5 (26.9)

No. of atomsCif — 2,135 2,094, 2,078NEDD8 — 634 635, 604Water — 376 318Others — 1 16

B-factors, Å2

Cif — 22 27, 24NEDD8 — 23 28, 36Water — 34 32Others 25 33

rmsdBond lengths, Å — 0.02 0.02Bond angles, ° — 2.05 1.95ESU (ML), Å — 0.08 0.10Ramachandran favored, % — 98.4 98.7Ramachandran outliers, % — 0 0

Values in parentheses correspond to the highest resolution bin. DLS, Diamond Light Source; ESU (ML), Estimated StandardUncertainty (Maxmium Likelihood).

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Structural studies of Cifs have shown they belong to a clan ofrelated enzymes that includes cysteine proteases, acetyltransferases,transglutaminases, and putative deamidases (36). Cifs are the firstmembers of this clan with deamidase activity to have theirstructures determined. The Cif/NEDD8 complexes describedhere allow us to explore how these enzymes have adapteda cysteine protease-like fold to perform a deamidation reactionand evolved to recognize their substrate.Both deamidation and proteolysis result in the hydrolysis of an

amide bond. Therefore, it is likely that the reaction catalyzed byCif will be fundamentally similar to the proteolysis reactioncatalyzed by cysteine proteases (37). In CifYp, residues thatcomprise the catalytic triad are Cys117, His173, and Gln193, withthe main-chain amides of Val116 and Leu196 contributing to anoxyanion hole. The adaptation of the cysteine protease-like foldto catalyze substrate-specific side-chain deamidation is unique tothese effectors and is most likely an example of divergent evo-lution from a common ancestor. However, what are the keyfeatures of Cif that deliver this unique adapted activity? Thesecan be explored by considering the two primary enzyme/substrateinterfaces formed by the Cif head and tail domains in the CifYp/NEDD8 and CifPl/NEDD8 complexes and their roles in pro-ductive substrate binding.

The head domain of Cifs supports the position of catalyticresidues within an active site cleft. The equivalent region incysteine proteases defines substrate specificity, recognizing resi-dues to both the N and C termini of the scissile bond, with thesubstrate presented in an extended conformation. In the CifYp/NEDD8 and CifPl/NEDD8 complexes, a prominent cleft inter-acts with residues on the α1/β3 loop of NEDD8 to the N terminusof Gln-40. Recognition of residues to the C terminus of Gln-40 isblocked by the occluding loop (23, 24), resulting in a very dif-ferent substrate-binding interface (Fig. 3D). A residue within thisloop, Leu196 (CifYp numbering), along with Val116, displacesthe C-terminal β-strand of NEDD8 from its native conformation,helping to position Gln40 in the active site (Fig. 3 D and E).Introduction of a charged residue in place of Val116 [CifYp(Val116Asp)] prevented Cif/NEDD8 interaction. Further, theOδ1 atom of Asp195 forms a hydrogen bond with the Nε2 atom ofNEDD8:Gln40. This interaction, along with the backbone amidesof Cys117 and Leu196 and the backbone carbonyl of Gly172,forms a pattern of hydrogen bond donor/acceptor residues thatensures the Nε2 atom, rather than the Oδ1 atom, of Gln40 isoriented toward His173 (Fig. 3F). This role is consistent with therelatively minor impact of the CifYp(Asp195Asn) mutation oncatalysis in vitro. In contrast, the equivalent mutation in CifEc

Fig. 3. Crystal structure of the CifYp/NEDD8 complex. (A) Cartoon representation of the CifYp/NEDD8 complex. CifYp is shown in green, with NEDD8 shown ingray-blue. The residues that comprise the interface between the two proteins are colored copper (for CifYp) and light blue (for NEDD8). (B) Space-fillingsurface representation of the complex. (C) Space-filling surface representation of CifYp and NEDD8, with NEDD8 rotated ∼180° and translated to reveal theinterface. Residues of CifYp that have been mutated in this study are shown in yellow and are labeled with a single-letter amino acid code. (D) Close-up viewof the CifYp (surface)/NEDD8 (cartoon) interface. In NEDD8, the substrate Gln40 is shown and the reoriented C terminus is colored gold. In Cif, residues of theoccluding loop are colored white. (E) Comparison of the free (Upper) and Cif-bound (Lower) conformations of NEDD8. Gln40 is shown, and the C-terminalresidues are colored as in D. (F) Interactions in the CifYp active site (green atoms in the cartoon) in complex with NEDD8 (gray-blue atoms in the cartoon).Hydrogen bonds (with distances) to the substrate Gln40 residue are shown as dashed lines.

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(Asp187Asn) did not trigger G2/M cell-cycle arrest in infectedHeLa cells (12), suggesting this subtle mutation may have func-tional relevance in the context of a cellular environment.The tail domains of Cifs bear no resemblance to the struc-

turally equivalent region in cysteine proteases and appear to bean adaptation evolved to interact with NEDD8/ubiquitin sub-strates. Mutation of a Leu106 in CifYp (a residue that directlycontacts NEDD8) to a Glu severely impairs formation of theCifYp(Cys117Ala)/NEDD8 complex, catalysis in vitro, and ac-tivity in vivo. The CifYp(Val104Glu) mutation, in a residue thatdoes not contact NEDD8, still binds NEDD8 to WT levels. Atthe base of the tail domain, Asp66 (CifYp) forms hydrogen-bonding interactions with both Thr7 and Thr9 of NEDD8; theadjacent Asp67 residue is oriented away from the substrate.Neither the CifYp(Asp66Ala) nor CifYp(Asp67Ala) mutationrevealed any significant effect on NEDD8 binding in vitro.Binding of NEDD8 to CifYp(Asp66Arg) was still observed butwas significantly lower than WT. Inspection of the CifYp/NEDD8structure suggests an Arg side chain could be accommodated atthis position in the complex. A double mutant of CifEc(Asp58Ala/Asp59Ala) lost the ability to deamidate NEDD8, asassayed by nPAGE (9). Because structural data for this region ofCifEc are not currently available, the potential impact of thisdouble mutation on the structure and how this might affectfunction (and/or enzyme stability) are difficult to assess.No single alanine substitution variant we made in CifYp sig-

nificantly affected binding to NEDD8 in vitro, suggesting thatnone of the interactions probed are, in their own right, critical tobinding. However, the introduction of bulky/charged sub-stitutions in key interface amino acids all resulted in disruptionof the interaction. In the case of CifYp(Leu106Glu), we also showthat this mutation affects catalysis in vitro and activity in vivo,and predict that the other bulky/charged substitutions will resultin the same effects, although this remains to be tested. Giventhese observations, as well as the extensive buried surface formedbetween Cif and NEDD8, we conclude that shape complemen-tarity is a key feature governing the interaction betweenthese proteins.In this study, we have mainly focused on the Cif/NEDD8 in-

teraction. However, Cifs can also catalyze the deamidation ofGln40 in ubiquitin. In vitro, we observe a preference of CifYp for

NEDD8 over ubiquitin, similar to that seen for CifBp (9).Modeling ubiquitin on the structure of NEDD8 in the CifYp/NEDD8 complex revealed only three positions at the interfacethat are different between these proteins: 31 (NEDD8:Glu/ubquitin:Gln), 39 (Gln/Asp), and 72 (Ala/Arg). The residue atposition 72 of NEDD8/ubiquitin is known to be a key de-terminant of specificity in both deneddylation (38) and E1-specificity (39) pathways. However, as supported by the structureand activity work presented here, all these changes can be ac-commodated in the CifYp/NEDD8 complex. Similarly, dockingthe structure of CifBp on the CifYp/NEDD8 complex does notsuggest any strong selective pressure for one or another sub-strate. Unlike CifYp and CifBp, CifEc is reported to have an∼1,000-fold preference for NEDD8 over ubiquitin (9). One hy-pothesis that could explain this differential activity is the lack ofconservation in amino acids of Cif (CifYp:Ile259, CifBp:His234,CifEc:Gln251, and CifPl:Lys269; Fig. 1) that contributes to therecognition of the residue preceding Gln40 in the substrate(Gln39 in NEDD8 and Asp39 in ubiquitin). Second, CifEc has

Fig. 4. Comparison of the overall structure of CifPl and NEDD8 in theiruncomplexed states and in the CifPl/NEDD8 complex. The structures of CifPland NEDD8 as found in the complex are shown in wheat and gray-blue.Those of the overlaid uncomplexed proteins are shown in cyan and magenta.

Fig. 5. CifYp and NEDD8 binding monitored using ITC. (A) Example ofbinding isotherm and associated fit for the CifYp(Cys117Ala)/NEDD8 in-teraction. (B) Bar chart representation of the dissociation constant betweenCifYp(Cys117Ala)/NEDD8, and additional variants as labeled, derived from fitsto the ITC curves (Table S6).

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a Ser residue at the position of an otherwise conserved Val(Val116 in CifYp), which is implicated in the reorientation of theC-terminal β-strand of NEDD8, although how this could con-tribute to substrate selectivity is not clear. Although acknowl-edging that the published structure of CifEc does not include thetail domain, these are two differences that may contribute toCifEc’s NEDD8 selectivity.Despite recent advances in defining the activity of Cifs, many

questions remain. First, does Cif deamidate “free” NEDD8 orNEDD8 already conjugated to CRLs? An overlay of NEDD8 inthe Cif/NEDD8 complexes with neddylated Cul5ctd-Rbx1 (16)shows it is unlikely that Cif can interact with neddylated cullins ina catalytically competent orientation (because of a significantclash), suggesting that Cifs deamidate NEDD8 before conjuga-tion. Further, when CifBp is overexpressed in HEK293T cellswith Cullin1, only very weak interaction is observed (22). Second,what is the precise mechanism by which NEDD8:Gln40 deami-dation leads to stabilization of neddylated CRLs? A recent studyconcluded that Cif-mediated NEDD8 deamidation inhibits CRLdeneddylation by the COP9 signalosome (CSN) in vivo (22). Thisstudy proposed two mechanisms by which this could be achieved:(i) Deamidation of Gln40 prevents NEDD8-induced conforma-tional changes in CRLs that are important for recognition byCSN, or (ii) deamidation of Gln40 directly inhibits recruitmentof CSN to neddylated CRLs. The latter supports hypotheses thatdeamidated ubiquitin impairs ubiquitination pathways, becausethe Gln40 side chain is involved in interaction with E3 ligases (9,40). It is difficult to see how Gln40 deamidation could alter thestructure of neddylated CRLs; this residue is presented to bulksolvent in the neddylated Cul5ctd-Rbx1 structure [Protein Data

Bank (PDB) ID code 3DQV] (16). Importantly, however, Gln40is positioned adjacent to the thioester link between the C ter-minus of NEDD8 and Lys724 of Cul5ctd (separated by only6.1 Å). Therefore, it is more likely that Cif-mediated deamida-tion of Gln40 directly interferes with CSN binding to neddylatedCRLs, although this remains to be verified experimentally. Fi-nally, what is the benefit to the pathogen of interfering with CRLactivity? Perhaps the resulting inhibition of the cell cycle delaysepithelial cell renewal at the site of infection, favoring coloni-zation, or reduced CRL activity augments the activity of othercodelivered effectors, enhancing the virulence of the pathogen.By targeting CRLs, Cifs could interfere with the stability ofhundreds of substrates, suggesting that this effector may modu-late many critical host cell functions, including cell proliferation,apoptosis, differentiation, and immune responses (7).Cifs are part of a growing number of microbial products that

interfere with ubiquitination pathways in eukaryotic cells. Futurestudies will undoubtedly use Cifs not only to address the role ofthese pathways in host/pathogen interactions but as tools todissect the role of such pathways in other aspects of cell biology.

Materials and MethodsCloning of Cifs and NEDD8 for Protein Production. The cloning of CifYp, CifBp,CifEc, and CifPl has been described previously (10, 41). In this study, wegenerated unique constructs for expression of CifBp and CifYp using existingvectors as templates. For CifBp, we used primers 5′-AAGTTCTGTTTCA-GGGCCCGatgataacgccgatcatttcatcg-3′ (Forward) and 5′-ATGGTCTAGAAA-GCTTTAgccaaggccggcgacgtattgtgc-3′ (Reverse) to amplify the regionencoding the full-length protein (23). These primers included DNA sequen-ces (capitalized letters) that enabled recombination-based cloning into thepOPIN-F expression vector using published procedures (42).

Fig. 6. Substrate deamidation by CifYp (and selected variants) monitoredusing nPAGE. (A) nPAGE analysis of NEDD8 deamidation by CifYp. C/A,CifYp(Cys117Ala); D/N, CifYp(Asp195Asn); L/E, CifYp(Leu106Glu). (B) nPAGEanalysis of ubiquitin deamidation by CifYp. (C) Quantification of CifYp dea-midase activity against NEDD8 and ubiquitin from enzyme-titration experi-ments (Fig. S5). CifYp (□), CifYp(Asp195Asn) (♢), CifYp(Leu106Glu) (△), andCifYp(Cys117Ala) (○), all with a solid line and with NEDD8 as the substrate,are shown. CifYp with ubiquitin (■), with a dashed line, is also shown.

Fig. 7. Effects of CifYp and variants on the cell-cycle arrest marker p21. (A)p21 accumulates in the presence of WT (wt) CifEc and CifYp but not in theactive site Cys mutants (C/A) or the CifYp(Leu106Glu) mutant (L/E). p21, actin,and His-tagged proteins were probed with appropriate antibodies. (B)Chemiluminescence signals of p21 and actin shown in A from six [or 3 forCifYp(Leu106Glu)] independent experiments were quantified. The averagesof p21 level are represented and expressed as ±SEM. Statistical differencesbetween experimental groups were determined using one-way ANOVA withthe Bonferroni multiple comparison posttest. **P < 0.01; ***P < 0.001.

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To produce recombinant CifYp in a soluble form, we generated anN-terminally truncated construct of this protein using primers 5′-AAGTTCT-GTTTCAGGGCCCGgtttcacattccataaataacccttcg-3′ (Forward) and 5′-ATGGTC-TAGAAAGCTTTAattacagtgagttttaatgattgacatattg-3′ (Reverse) that ampli-fied residues 33–290 of the native sequence. The resulting PCR product wascloned into pOPIN-F as above. We also generated the CifYp(Cys117Ala) mu-tant in pOPIN-F using the same primers and a template DNA that alreadyincluded this mutation.

DNA encoding full-length NEDD8 was amplified from a synthetic templateobtained from Geneart and PCR primers 5′-AGGAGATATACCATGctaatta-aagtgaagacgctgaccggaaag-3′ (Forward) and 5′-GTGATGGTGATGTTTctgcct-aagaccacctcctcctctcagagc-3′ (Reverse). The resulting product was cloned intopOPIN-E (42), generating a C-terminal 6× His-tag.

The DNA sequence of all constructs was verified by sequencing.

Mutagenesis of CifYp and NEDD8. All the additional mutants established in theCifYp(Cys117Ala) background were produced by Genscript, using the pOPIN-F:CifYp(Cys117Ala) plasmid as a template. The Gln40Glu and Gln40Alamutants of NEDD8 were also produced by Genscript, using the pOPIN-E:NEDD8 plasmid as a template.

Expression and Purification of Cif Proteins. CifYp and CifBp were expressedfrom pOPIN-F, and CifEc and CifPl were expressed from pET28a-based plas-mids. Soluble CifYp was obtained using E. coli SoluBL21 (Gelantis); expressionof all other Cifs used E. coli BL21(DE3). Bacterial cultures were grown in LBbroth at 37 °C, supplemented with carbenicillin (pOPIN-F, 100 mg/L) orkanamycin (pET28a, 30 mg/L), before induction with 1 mM isopropyl-β-D-thiogalactopyranoside and overnight growth at 16 °C. Cells were pelleted at5,000 × g, frozen, and stored at −80 °C until ready for purification. Thawedcell pellets were resuspended in 50 mM Hepes (pH 7.8), 300 mM NaCl, and25 mM imidazole before being lysed by sonication. Unbroken cells and de-bris were cleared by centrifugation at 30,000 × g for 25 min. Cleared lysateswere loaded onto preequilibrated Ni2+-immobilized metal ion affinitychromatography (IMAC) columns; washed extensively in load buffer; andstep-eluted in 50 mM Hepes (pH 7.8), 300 mM NaCl, and 250 mM imidazole.The eluate was directly loaded onto a Hi-Load 26/60 Superdex 75 gel fil-tration column (GE Healthcare); equilibrated; and run in 20 mM Hepes(pH 7.5), 150 mM NaCl, and 1 mM DTT (DTT was excluded from the buffer forpurification of proteins used in ITC experiments). Proteins were concen-trated by ultrafiltration to between 350 μM and 950 μM before aliquotingand flash-freezing in liquid nitrogen. All protein aliquots were stored frozenuntil required for experiments.

Preparation of NEDD8 and Ubiquitin. NEDD8 was expressed from pOPIN-Eusing E. coli SoluBL21. Purification proceeded as described for Cifs, and DTTwas not included in the gel filtration buffer.

Ubiquitin was purchased from Sigma (bovine, identical sequence to humanubiquitin) and redissolved in 20 mMHepes (pH 7.5) and 150 mMNaCl for use.

Purification and Crystallization of Cif/NEDD8 Complexes. To generate Cif/NEDD8 complexes for purification and subsequent crystallization, weadopted a “cosplitting” approach, where pelleted bacterial cells previouslyinduced to express individual Cif proteins were mixed and resuspended withpelleted cells containing expressed NEDD8. The mixture of resuspended cellswas then sonicated, and proteins were purified as above, retaining elutionpeaks that contain complexes as judged by SDS/PAGE. We attempted tosaturate Cif with NEDD8 by adding excess culture expressing this secondprotein. Saturation was evident from the presence of a large peak corre-sponding to free NEDD8 in the gel filtration profile at a high retentionvolume and the absence of a peak or shoulder at elution volumes thatwould correspond to free Cifs. For CifBp and CifYp, we also produced samplesin which the N-terminal His-tag was removed using 3C protease (sampleswere concentrated to ∼700 μM and treated with 0.01 mg/mL 3C proteaseovernight at room temperature).

Crystallization of Cif/NEDD8 Complexes. We produced Cif/NEDD8 complexesusing CifYp, CifBp, CifEc, and CifPl with various catalytic site mutants. Extensivescreening of crystallization conditions for these complexes using roboticsetups identified crystals for CifPl(Cys123Ser)/NEDD8 and CifYp(Cys117Ala)/NEDD8. The latter was from a sample treated with 3C protease. CifPl(Cys123Ser)/NEDD8 crystals were confirmed to comprise both proteins usingMS. Diffraction quality crystals of CifPl(Cys123Ser)/NEDD8 were obtainedfrom 20% (vol/vol) PEG 4000, 200 mM sodium acetate, and 100 mM Mes (pH6.7) using a 96-well sitting drop plate and a drop composed of 0.35 μL ofprotein solution [∼600 μM complex in 20 mM Hepes (pH 7.5), 150 mM NaCl]

and 0.65 μL of the crystallization reagent. Diffraction quality crystals of CifYp(Cys117Ala)/NEDD8 crystals were grown from 2.2 M sodium malonate, 44mM Bis-Tris propane (pH 7), 66 mM Bis-Tris propane (pH 8) using a 1-μL sittingdrop composed of 0.35 μL of the protein sample [∼500 μM complex, 20 mMHepes (pH 7.5), 150 mM NaCl], and 0.65 μL of the crystallization reagent.

X-Ray Data Collection, Structure Solution, Refinement, and Validation. Crystalsof CifPl(Cys123Ser)/NEDD8 and CifYp(Cys117Ala)/NEDD8 were frozen ina cryoprotectant solution composed of 75% of the mother liquor (takendirectly from the crystallization plate) and 25% ethylene glycol. All X-raydata were processed with iMosflm (43) and scaled with Scala (44), asimplemented in the CCP4 suite (45). X-ray data collection statistics are givenin Table 1.

The structure of CifPl(Cys123Ser)/NEDD8 was solved by molecular re-placement with data collected on a Rigaku RU-H3RHB generator/Mar345detector and MolRep (45), using search models derived from CifPl andNEDD8 [PDB ID codes 3GQY (CifPl) and 1NDD (NEDD8)]. Iterative cycles ofrefinement with Refmac5 (45) and manual rebuilding with Coot (46) gen-erated a model that was fitted to high-resolution data (from the samecrystal) obtained at beamline I02 of the Diamond Light Source (Oxford,United Kingdom). Refinement/rebuilding cycles as above generated the finalmodel. Anisotropic B-factors and alternate conformations were added to-ward the end of refinement.

The structure of CifYp(Cys117Ala)/NEDD8 was solved by molecular re-placement with data collected at beamline I24 of the Diamond LightSource and MolRep. An edited version of the CifPl(Cys123Ser)/NEDD8 wasused as a model (essentially polyalanine traces were generated). Theresulting phases were modified by Parrot (47); Buccaneer (47) was used torebuild the structure with the correct sequence. Iterative cycles of re-finement with Refmac5 and manual rebuilding with Coot generated thefinal model.

Structure validation used Coot and Molprobity (48). A selection of re-finement and validation statistics is given in Table 1. Structure-basedoverlays were calculated using secondary-structure matching algorithmsas implemented in Superpose from the CCP4 suite (25, 45). The coor-dinates and structure factors for the CifYp/NEDD8 and CifPl/NEDD8 com-plexes have been submitted to the PDB with ID codes 4F8C (CifYp/NEDD8)and 4FBJ (CifPl/NEDD8).

Protein/Protein Interaction Studies (ITC). Cifs and NEDD8 were both preparedin 20 mM Hepes (pH 7.5) and 150 mM NaCl, and were diluted consistentlyinto the same buffer. All ITC experiments were performed with a MicroCal205 calorimeter in high gain mode. In a typical experiment, the calorimetrycell was filled with 205 μL of Cif at ∼100 μM before making sequentialinjections of ∼1,200 μM NEDD8 from the syringe up to a final cumulativeinjection volume of 38 μL. Experiments were conducted at 25 °C, withtypical injections of between 1.0 μL and 2.0 μL at 180-s intervals. Twocontrol experiments were performed to ensure that the heat of dilution ofNEDD8 or Cif would not be problematic under these experimental con-ditions; these involved direct injections of NEDD8 into buffer and injectionsof buffer into a solution of Cif. Binding isotherms were fitted to the in-tegrated calorimetric data using Origin (Microcal). Each binding experimentwas performed at least three times (except for the Cys117Ala/Asp66Argvariant, which was performed twice), with the mean and SD calculated foreach variant.

nPAGE to Monitor Enzyme Activity. A total of 700 pmol of NEDD8, NEDD8(Q40A), and NEDD8(Q40E) was incubated with 0.1 pmol of CifYp (or variants)for 30 min at 30 °C. Total reaction volume was 10 μL. The reaction mixturewas chilled on ice, diluted with 1:1 loading buffer [50 mM glycine (pH 10.0),20% (vol/vol) glycerol], and 10 μL was loaded onto 12.5% nPAGE gelsbuffered with 50 mM glycine at pH 10.0 (gels were run with the samebuffer). Therefore, 350 pmol of substrate was loaded on the gel. Proteinswere visualized with InstantBlue staining, and the bands quantified usingImageJ (National Institutes of Health). The same protocol was used for theubiquitin assays, except 1.0 pmol of CifYp (or variants) was incubated with700 pmol of substrate and gels were stained with Coomassie Blue.

For the titration experiments, the range of enzyme quantities used isshown in Fig. 6C and Fig. S5.

Lipofection Experiments. Lipofection assays of purified Cif proteins wereconducted essentially as previously described (10). HeLa cells (CCL-2; Amer-ican Type Culture Collection) were cultured in DMEM (Invitrogen) supple-mented with 10% FCS (Eurobio) and 80 mg/L gentamicin at 37 °C in a 5%CO2 atmosphere. For lipofection assays, 80 μL of purified CifYp, CifEc (500 μg/

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mL), or PBS as a negative control was added to one Bio-PORTER tube(Genlantis) and resuspended in 420 μL of DMEM. The samples were added tothe HeLa cells in six-well plates and incubated for 4 h before being replacedby fresh growth medium and further incubated for 20 h.

For Western blot analyses, ∼6 × 105 cells were lysed in 80 μL of SDS/PAGEsample buffer, sonicated for 2 s to shear DNA, and then boiled for 5 min.Protein samples were resolved on either 10% or 4–12% NuPage gradientgels (Invitrogen) and blotted on PVDF membranes. Membranes wereblocked in 10 mM Tris (pH 7.8), 150 mM NaCl, 0.1% Tween 20, and 5%nonfat dry milk, and then probed with primary antibody (0.5 mg/mL−1) inthe same buffer. Primary antibodies used were anti-actin (ICN), anti-p21 (CellSignaling Technology), and anti-histidine (Qiagen). Bound antibodies were

visualized with HRP-conjugated secondary antibody. Acquisitions were per-formed with a Molecular Imager ChemiDoc XRS system (Bio-Rad). Proteinlevels were quantified with Quantity One software (Bio-Rad) and normal-ized with the actin level.

ACKNOWLEDGMENTS. We thank Dr. Jean-Philippe Nougayrède (Toulouse)and all the M.J.B. group for discussions. We acknowledge the staff ofthe Diamond Light Source synchrotron radiation source for access todata collection facilities. This work was supported by Grant F008732 of theBiotechnology and Biological Sciences Research Council, United Kingdom (toM.J.B.), a Royal Society (United Kingdom) University Research Fellowship (toM.J.B.), and the Ligue Nationale Contre le Cancer (F.T.).

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