The Molecular Basis of Ubiquitin-Like Protein NEDD8 Deamidation by the Bacterial Effector Protein Cif

The molecular basis of ubiquitin-like protein NEDD8 deamidation by the bacterial effector protein Cif

Allister 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-31076 Toulouse, France; cInstitut National de la Recherche Agronomique, USC 1043, F-31300 Toulouse, France; dInstitut National de la Santé et de la Recherche Médicale, 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 de Bactériologie-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 translocated uitinylation and targeting to the 26S proteasome (13). In this effector proteins, found in diverse pathogenic bacteria, that system, ubiquitin molecules are covalently attached to proteins interfere with the host cell cycle by catalyzing the deamidation destined for destruction by the concerted action of an E1-E2-E3 of a specific glutamine residue (Gln40) in NEDD8 and the related enzyme cascade, with substrate specificity defined by E3 ligases protein ubiquitin. This modification prevents recycling of neddylated (14). The largest family of E3 ligases is the cullin RING E3 cullin-RING ligases, leading to stabilization of various cullin-RING li- ubiquitin ligases (CRLs) (15). As befitting their critical role in gase targets, and also prevents polyubiquitin chain formation. Here, many cellular processes, the activities of CRLs are tightly regu- we report the crystal structures of two Cif/NEDD8 complexes, related. One mechanism for CRL activation is through conjugation vealing a conserved molecular interface that defines enzyme/sub- of the ubiquitin-like molecule NEDD8 (neural precursor cell strate recognition. Mutation of residues forming the interface expressed, developmentally down-regulated 8) to the cullin suggests that shape complementarity, rather than specific individ- subunit (neddylation) (16, 17), stimulating substrate ubiquitina- ual interactions, is a critical feature for complex formation. We tion. Importantly, cycling of CRLs between neddylated and show that Cifs from diverse bacteria bind NEDD8 in vitro and con- deneddylated forms is required for full activity (18, 19). clude that they will all interact with their substrates in the same Cif from enteropathogenic Escherichia coli (CifEc) interacts way. The “occluding loop” in Cif gates access to Gln40 by forcing with NEDD8 in both yeast two-hybrid assays (20) and Proto- a conformational change in the C terminus of NEDD8. We used Array analysis (21). CifEc also colocalizes with NEDD8 in the native PAGE to follow the activity of Cif from the human pathogen nuclei of HeLa cells (20) and specifically binds to neddylated Yersinia pseudotuberculosis and selected variants, and the posi- CRLs, but not to the unmodified proteins, in immunoprecipita- tion of Gln40 in the active site has allowed us to propose a catalytic tion assays (20). Significantly, CifEc was shown to inhibit the E3 mechanism for these enzymes. ligase activity of neddylated cullins (7, 9, 20, 21). Cif activity is correlated with accumulation of CRLs in their neddylated forms bacterial pathogenesis | cyclomodulins | host cell manipulation | structural (22), preventing the neddylation/deneddylation cycle and locking biology | type III secreted effector proteins CRLs in a neddylated but inactive form. This leads to stabilization of numerous CRL targets in cells, which presumably triggers any pathogenic Gram-negative bacteria use a type III the downstream cytopathic phenotype. Photorhabdus Msecretion (T3S) system to translocate effector proteins Structural studies of CifEc,CifBp,andCiffrom into target cells (1). Once inside the host cell, effectors act to luminescens (CifPl) (12, 23, 24) revealed a common fold, despite subvert and/or otherwise manipulate vital cellular systems and sharing low overall sequence identity. The proteins comprise represent a key virulence strategy for these pathogens (2). Type a head-and-tail domain structure reminiscent of a comma or III secreted effectors (T3SEs) can encode a wide range of dif- apostrophe. The C-terminal head domain comprises a cysteine ferent enzymatic activities (3). Because of the generally low protease-like fold and contains a conserved Cys-His-Gln catalytic amino acid sequence conservation of effectors to proteins of triad. Regions of the N-terminal tail domain are important for Cif known function, these activities are often only identified function, and it has been hypothesized that they contribute to through structural studies. substrate recognition (12, 20). A fundamental advance in un- In the past decade, progression of the host cell cycle has derstanding the mechanism by which Cifs inhibit CRL activity emerged as one cellular system targeted by multiple T3SEs from emerged when these effectors were shown to possess a specific – diverse pathogens (4 6). The cycle inhibiting factors (Cifs) deamidase activity (9). CifBp and CifEc both catalyze the deami- comprise a family of T3SEs from animal pathogens and insect dation of Gln40 in NEDD8 (converting this residue to a Glu). symbionts (7) that induce a cytopathic phenotype in host cells CifBp also efficiently deamidates Gln40 of ubiquitin. Ectopic ex- that includes cell-cycle arrest at the G2/M or G1/S transition (6, pression of a NEDD8(Gln40Glu) mutant in HeLa cells led to 8–12). It has been suggested that during the infection process, stabilization of CRL substrates and generated an equivalent effect restriction of the host cell cycle might delay epithelial cell renewal and favor gut colonization (7). Recently, regulation of

ubiquitin-mediated proteolysis has been implicated in the Author contributions: A.C. and M.J.B. designed research; A.C., R.K.H., and F.T. performed mechanism of Cif-induced cell-cycle arrest (7). Analysis of host research; A.C., R.K.H., F.T., E.O., and M.J.B. analyzed data; and A.C. and M.J.B. wrote cell proteins regulating cell-cycle checkpoints revealed accumu- the paper. Waf1/Cip1 lation of cyclin-dependent kinase inhibitors p21 and The authors declare no conﬂict of interest. Kip2 p27 in response to Cifs (10, 11); these proteins are usually This article is a PNAS Direct Submission. degraded by ubiquitin-mediated proteolysis. Further, ubiquitin- Data deposition: Atomic coordinates and structure factors reported in this paper have mediated proteolysis of GFP reporters expressed in HeLa cells been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 4F8C–4FBJ). was blocked following delivery of Cif from Burkholderia pseu- 1To whom correspondence should be addressed. E-mail: mark.banﬁ[email protected]. domallei (CifBp) (9). See Author Summary on page 10755 (volume 109, number 27). One mechanism for managing eukaryotic cell-cycle pro- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. gression is timed degradation of key regulators through ubiq- 1073/pnas.1112107109/-/DCSupplemental.

E1830–E1838 | PNAS | Published online June 12, 2012 www.pnas.org/cgi/doi/10.1073/pnas.1112107109 Downloaded by guest on September 24, 2021 PNAS PLUS to that exhibited by Enteropathogenic E. coli (EPEC) infection plexes in the CifYp(Cys117Ala)/NEDD8 crystal and 1.41/1.47 Å (9). This provides strong evidence that Cif deamidase activity to- between the CifPl(Cys123Ser)/NEDD8 and the two CifYp ward NEDD8-Gln40 is necessary and sufficient for the Cif-me- (Cys117Ala)/NEDD8 complexes (320, 279, and 284 equivalent diated cytopathic phenotype. Cα atoms considered). These structures most likely represent The purpose of this study was to investigate the interaction a substrate-binding mode that is conserved across the Cif family. between Cifs and NEDD8, both biochemically and structurally, A cartoon representation of the CifYp(Cys117Ala)/NEDD8 and also to probe the mechanism of deamidation. Here, we complex (henceforth CifYp/NEDD8) is shown in Fig. 3A. report the crystal structures of two Cif/NEDD8 complexes, one including Cif from the human pathogen Yersinia pseudotuber- Crystal Structure of CifYp. The crystal structure of free CifYp has culosis (CifYp) and the second from the insect symbiont not been reported previously. Although in complex with P. luminescens. The structure of CifYp has not been determined NEDD8, the structure of CifYp is very similar to other Cifs. It before. The two complexes share a common mode of binding overlays on CifBp (23), CifPl (23), and the truncated structure of with interactions arising from both the head and tail domains. CifEc (24) with rmsds (25) of 1.69 Å, 1.72 Å, and 1.20 Å (231, The Gln40 substrate residue of NEDD8 extends into the cata- 229, and 167 equivalent Cα atoms considered). The catalytic triad lytic site of the Cifs. We also show that other members of the Cif residues Cys117, His173, and Gln193 (CifYp numbering) occupy family bind to NEDD8 and suggest that our structures are essentially equivalent positions in all structures. a model for all Cif/NEDD8 complexes. Using site-directed mutagenesis of CifYp, we have probed the enzyme/substrate- An Extensive Binding Interface Is Formed Between CifYp/Pl and NEDD8. binding interface and hypothesize that overall shape comple- Structures of uncomplexed Cifs have been described as compris- mentarity rather than any specific individual interaction is the ing a head-and-tail domain structure reminiscent of a comma driving force behind complex formation. Finally, we have also or apostrophe (23). The structures of CifYp/NEDD8 and CifPl investigated the catalysis of NEDD8 and ubiquitin deamidation (Cys123Ser)/NEDD8 independently show that both the head by CifYp. This work has allowed us to propose a mechanism for domain [which comprises residues Val116/122 (CifYp/CifPl)tothe the activity of Cifs that ultimately leads to inhibition of the host C terminus] and tail domain [which comprises residues from the ubiquitin-dependent proteasomal degradation pathway and the N terminus to Pro115/121 (CifYp/CifPl)] make significant con- cytopathic phenotype. tributions to the NEDD8 binding interface (Fig. 3 A–D). This interface buries 1508.5 Å2 of the NEDD8 solvent-accessible sur- Results face area in the CifYp/NEDD8 complex, equivalent to 31.1% Cifs Crystallize in a 1:1 Complex with NEDD8. To define how the Cif of the total [1,376.1 Å2 (29.2% of the total) is buried in the family of effectors (sequence alignment is shown in Fig. 1) CifPl(Cys123Ser)/NEDD8 complex]. Both the CifYp/NEDD8 and engages their substrate and catalyzes the deamidation of CifPl(Cys123Ser)/NEDD8 interfaces have a complexation signifi- NEDD8, we attempted to crystallize a variety of Cif/NEDD8 cance score of 1.00 as defined by Protein Interfaces, Surfaces and complexes (details of gene cloning and protein expression are Assemblies (PISA) (26). NEDD8 residues that form the interface Materials and Methods provided in ). We obtained diffraction- with Cif reside on the loop between β1 and β2 (Lys6–Lys11), α1 quality crystals of catalytic site mutants CifPl(Cys123Ser) and and β3 (Glu31–Arg42, which contains the substrate Gln40 resi- CifYp(Cys117Ala) in complex with NEDD8 following copur- due), β3 and β4 (Ile44–Gly47), and the C-terminal β-strand ification of the enzyme and substrate (Materials and Methods, (Val66–Arg74). These four regions form interactions with the tail, Fig. 2, Fig. S1, and Table S1). Both structures were solved by head, tail, and tail-and-head domains of Cif, respectively. Full molecular replacement using uncomplexed Cifs and NEDD8 as details of the residues contributing to the interfaces and the search models. X-ray data collection, refinement, and validation interactions they form are given in Tables S2–S5. statistics are given in Table 1. The CifPl(Cys123Ser)/NEDD8 and CifYp(Cys117Ala)/NEDD8 crystal structures comprise one and Changes in Conformation on Complex Formation. The availability of two 1:1 complexes of Cif and NEDD8 in the asymmetrical unit, the uncomplexed CifPl (23) and NEDD8 (27) structures, along- BIOCHEMISTRY respectively. Each of these complexes shows very similar overall side the CifPl(Cys123Ser)/NEDD8 complex (henceforth CifPl/ arrangements with rmsds (25) of 0.59 Å between the two com- 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 in

red (tail domain) and green (head domain) are residues in CifYp that were mutated to investigate either NEDD8 binding and/or catalysis. The residue that may be involved in NEDD8/ubiquitin selectivity is shown in yellow. Asterisks denote positions of identical residues.

Crow et al. PNAS | Published online June 12, 2012 | E1831 Downloaded by guest on September 24, 2021 dependently confirmed the 1:1 binding stoichiometry previously observed by gel filtration, further supporting the validity of the interaction observed in the crystals. The interaction between CifYp(Cys117Ala) and NEDD8 was also analyzed using intrinsic tryptophan fluorescence (SI Text and Figs. S2 and S3).

Targeted Mutagenesis of the Cif/NEDD8 Binding Interface Disrupts the Interaction. Having quantified the interaction between CifYp(Cys117Ala) and NEDD8, we investigated the importance of interfacing residues identified in the CifYp/NEDD8 structure. First, we constructed five independent alanine substitution mutants in residues that we hypothesized would form important interactions with NEDD8 (CifYp tail domain residues Asp66 and Asp67 and CifYp head domain residues Asn122, Asn167, and Leu196; Fig. 3 B and C and Tables S2– S5). All but CifYp(Asn122Ala) were expressed and purified, and displayed comparable chemical unfolding/refolding profiles to WT protein; therefore, these mutations do not significantly affect stability (Fig. S4 and Table S7). Somewhat surprisingly, each of these mutants, which included examples of removing intermolecular hydrogen bonds and hydrophobic interactions, gave only marginal decreases in the affinities as measured by ITC (Fig. 5B and Table S6); similar results were obtained using fluorescence-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 interface

Fig. 2. Gel filtration enables purification of a 1:1 complex of CifYp and positions. Two of these mutations [Asp67Arg and Val104Glu fi fi NEDD8. (A) Gel ltration traces derived from puri cations of CifYp, NEDD8, (tail domain)] were designed as controls and targeted residues and the complex. Details of the elution volumes are given in Table S1. mAU, that did not closely associate with NEDD8 in the structure, and milli-absorbance units. (B) SDS/PAGE analysis of fractions collected across the four [Asp66Arg and Leu106Glu (tail domain) and Val116Asp elution peaks corresponding to the CifYp/NEDD8 complex and excess NEDD8 and Gly118Thr (head domain)] were predicted to compromise (from the same gel filtration experiment). CifYp/NEDD8 interaction (Fig. 3C). All variant proteins except Asp67Arg were expressed and purified, and displayed comparable fi tional changes occurring during complex formation. Overall, the chemical unfolding/refolding pro les to WT protein (Table S7). Although the affinity of the interaction between CifYp(Val104Glu) structures of CifPl and NEDD8 are very similar in their unbound and bound forms (rmsds of 0.86 Å and 0.62 Å, using 247 and 71 and NEDD8 was equivalent to WT, CifYp(Leu106Glu), CifYp (Val116Asp), and Cif (Gly118Thr) were each severely compro- equivalent Cα atoms, respectively) (25) (Fig. 4). The only notable Yp mised in NEDD8 binding as measured by ITC (Fig. 5B and change in CifPl on binding NEDD8 is a slight reorientation at the fl tip of the tail domain (residues Ile94–Tyr116) that bends toward Table S6); similar results were obtained using uorescence (Fig. S2 and Table S6). CifYp(Asp66Arg) showed an intermediate the substrate protein (maximum Cα shift of 3.4 Å for Glu100). In effect but was still significantly impaired in binding to NEDD8. NEDD8, the C-terminal five residues (Val70–Arg74) undergo fi a signi cant shift on binding CifPl (9.7 Å for the Cα of Arg74) NEDD8 Residue Glutamine 40 Occupies the Cif Active Site. The that breaks β-sheet hydrogen-bonding interactions and displaces α β structures of CifYp/NEDD8 and CifPl/NEDD8 revealed that the the C-terminal residues away from the 1/ 3 loop (Fig. 4). The substrate NEDD8 residue Gln40 projects from the loop between α1/β3 loop contains the substrate NEDD8:Gln40 residue, and the α1 and β3 into the Cif active site (Fig. 3F). In the CifPl/NEDD8 ε displacement of the C terminus appears critical for positioning complex, the N 2 atom of NEDD8:Gln40 forms a hydrogen bond D–F this residue in the Cif active site (Fig. 3 ). CifPl residues to the OH group of Ser123 (the mutated catalytic residue); it is Val122 and Leu203 are the key residues that enable the dis- unlikely that this represents a catalytically competent orientation placement of this region. The same displacement of the C ter- for this side chain. In the CifYp/NEDD8 complex, the Cys117Ala minus of NEDD8 is observed in the CifYp/NEDD8 complex with mutation allows the side chain of NEDD8:Gln40 to lie directly Val116 and Leu196 substituting for Val122/Leu203 (Fig. 3 D–F). over what would be the catalytic center. We produced a model of This Val/Leu pair is conserved in all Cifs except CifEc, where the the WT CifYp/NEDD8 complex by mutating (in silico) Ala117 equivalent Val is a Ser residue. back to a Cys (Fig. 3F). In this model, the thiol group of Cys117 is well-positioned for nucleophilic attack of the NEDD8: fi δ Cif Proteins Bind NEDD8 in Solution. Using gel ltration, we showed Gln40C atom, which would initiate the deamidation reaction. that CifYp, CifEc, CifBp, and CifPl (all containing mutations in the From the structures of the complexes, we also identified ad- active site Cys) were able to bind NEDD8, with the increases in ditional Cif residues that may be relevant for the catalytic ac- apparent molecular mass on complex formation consistent with tivity. Foremost among these is Asp195 (CifYp numbering), a δ 1:1 binding (Fig. 2, Fig. S1, and Table S1). We then focused on residue conserved in all Cifs. The Asp195-O 1 atom forms a hy- ε2 the CifYp/NEDD8 interaction as a model system to probe the drogen bond with NEDD8:Gln40N (2.81 Å) in the CifYp/ properties of Cif/NEDD8 binding attributable to the availability NEDD8 complex (Fig. 3F). Other interactions in the CifYp/ of the crystal structure and this pathogen’s relevance for NEDD8 active site include hydrogen bonds between NEDD8: δ1 human disease. We quantified the interaction between CifYp Gln40O with the backbone amides of Cys117 (2.77 Å) and ε (Cys117Ala) and NEDD8 using isothermal titration calorimetry Leu196 (2.94 Å) and NEDD8:Gln40N 2 with the backbone car- (ITC). This experiment showed the affinity was in the sub- bonyl of Gly172 (3.13 Å). The roles of these residues in orienting micromolar range (Fig. 5 and Table S6). The ITC data in- the NEDD8(Gln40) side chain are discussed below.

E1832 | www.pnas.org/cgi/doi/10.1073/pnas.1112107109 Crow et al. Downloaded by guest on September 24, 2021 Table 1. X-ray data collection, reﬁnement, and validation statistics PNAS PLUS

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

Data collection

Space group P21 P21 P6322 Cell dimensions a, b, c; Å 40.7, 56.0, 67.6 40.7, 56.1, 67.6 125.4, 125.4, 169.9 α, 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) Reﬁnement Resolution, 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 atoms Cif — 2,135 2,094, 2,078 NEDD8 — 634 635, 604 Water — 376 318 Others — 116 B-factors, Å2 Cif — 22 27, 24 NEDD8 — 23 28, 36 Water — 34 32 Others 25 33 rmsd Bond lengths, Å — 0.02 0.02 Bond angles, ° — 2.05 1.95 ESU (ML), Å — 0.08 0.10 Ramachandran favored, % — 98.4 98.7 Ramachandran outliers, % — 00

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

NEDD8 and Ubiquitin Deamidation by CifYp. Using tryptic digest and ubiquitin (Fig. 6C and Fig. S5). These assays show that ﬁ ﬁ liquid chromatography MS, we con rmed that the only modi - NEDD8 is a better substrate for CifYp than ubiquitin (∼0.025 cation to NEDD8 from exposure to CifYp was the conversion of pmol of CifYp required for complete conversion of 350 pmol of Gln40 to a Glu. We then used native PAGE (nPAGE) to in- NEDD8 in the assay compared with ∼0.25 pmol needed for vestigate the deamidase activity of Cif (9, 28). This assay ena- Yp ubiquitin). This is consistent with previous studies of CifBp and

bles screening of selected structure-informed mutants for effects BIOCHEMISTRY CifEc that also show a preference for NEDD8 (9). To support on activity. Incubation of NEDD8 with CifYp results in a shift in these results, we also performed enzyme titration experiments electrophoretic mobility equivalent to NEDD8(Gln40Glu); no with NEDD8 and the Cif (Cys117Ala), Cif (Leu106Glu), and shift occurs for NEDD8(Gln40Ala) (Fig. 6A). Further, this shift Yp Yp Cif (Asp195Asn) variants (Fig. 6C and Fig. S5). is dependent on the catalytic residue Cys117 [incubation with Yp Cif (Cys117Ala) does not result in a shift] (Fig. 6A). Yp p21 Accumulates in HeLa Cell Culture in the Presence CifYp. Delivery We also tested the activity of the CifYp(Leu106Glu) and of purified Cif proteins to HeLa cells (CifEc, CifBp, and Cif from CifYp(Asp195Asn) variants using the nPAGE assay. The Photorhabdus species) results in the accumulation of cell-cycle Cif (Leu106Glu) mutation compromises the interaction of Yp regulators, including p21 and p27, with this activity dependent on Cif (Cys117Ala) with NEDD8, despite its position near the Yp the active site cysteine (10). We have shown that WT Cif also tip of the tail domain, distant from the active site. Consistent Yp stabilizes p21, although the effect was weaker than that observed with this, catalytic activity for the CifYp(Leu106Glu) variant is significantly reduced compared with WT (Fig. 6A). The struc- for CifEc (Fig. 7). Both CifYp(Cys117Ala), a catalytic site mutation, and CifYp(Leu106Glu), a tail domain mutation distant from tures of CifYp/NEDD8 and CifPl/NEDD8 suggested a putative the active site (that severely compromises CifYp/NEDD8 in- role for Asp195 (CifYp numbering) in catalysis. We found that this variant still retained a significant level of activity, suggesting teraction and catalysis in vitro), also prevent p21 accumulation in that an Asp at this position is not critical for function and is HeLa cells (Fig. 7). unlikely to act as a general acid/base in catalysis (Fig. 6A). Discussion We also tested the ability of CifYp, CifYp(Cys117Ala), and Pathogenic bacteria of both animals and plants have evolved CifYp(Leu106Glu) to deamidate ubiquitin (Fig. 6B). Similar to NEDD8, we observed an electrophoretic shift of ubiquitin in the mechanisms for directly modifying host cell targets through the presence of the WT enzyme but not with CifYp(Cys117Ala) delivery of enzymes by the T3S system. These enzymes encode or CifYp(Leu106Glu). a variety of activities (3). Hydrolytic activity is emerging as a key To obtain further details of CifYp’s deamidase activity, we mechanism used by T3SEs, with many examples of proteases performed enzyme titration experiments (9, 28) with NEDD8 (29–33) and phosphatases (34, 35) acting in host cells.

Crow et al. PNAS | Published online June 12, 2012 | E1833 Downloaded by guest on September 24, 2021 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 in gray-blue. The residues that comprise the interface between the two proteins are colored copper (for CifYp) and light blue (for NEDD8). (B) Space-ﬁlling surface representation of the complex. (C) Space-ﬁlling surface representation of CifYp and NEDD8, with NEDD8 rotated ∼180° and translated to reveal the interface. 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 view of 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 the occluding loop are colored white. (E) Comparison of the free (Upper) and Cif-bound (Lower) conformations of NEDD8. Gln40 is shown, and the C-terminal

residues 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.

Structural studies of Cifs have shown they belong to a clan of The head domain of Cifs supports the position of catalytic related enzymes that includes cysteine proteases, acetyltransferases, residues within an active site cleft. The equivalent region in transglutaminases, and putative deamidases (36). Cifs are the first cysteine proteases defines substrate specificity, recognizing resi- members of this clan with deamidase activity to have their dues to both the N and C termini of the scissile bond, with the structures determined. The Cif/NEDD8 complexes described substrate presented in an extended conformation. In the CifYp/ here allow us to explore how these enzymes have adapted NEDD8 and CifPl/NEDD8 complexes, a prominent cleft inter- a cysteine protease-like fold to perform a deamidation reaction acts with residues on the α1/β3 loop of NEDD8 to the N terminus and evolved to recognize their substrate. of Gln-40. Recognition of residues to the C terminus of Gln-40 is Both deamidation and proteolysis result in the hydrolysis of an blocked by the occluding loop (23, 24), resulting in a very dif- amide bond. Therefore, it is likely that the reaction catalyzed by ferent substrate-binding interface (Fig. 3D). A residue within this Cif will be fundamentally similar to the proteolysis reaction loop, Leu196 (CifYp numbering), along with Val116, displaces catalyzed by cysteine proteases (37). In CifYp, residues that the C-terminal β-strand of NEDD8 from its native conformation, comprise the catalytic triad are Cys117, His173, and Gln193, with helping to position Gln40 in the active site (Fig. 3 D and E). the main-chain amides of Val116 and Leu196 contributing to an Introduction of a charged residue in place of Val116 [CifYp oxyanion hole. The adaptation of the cysteine protease-like fold (Val116Asp)] prevented Cif/NEDD8 interaction. Further, the δ ε to catalyze substrate-specific side-chain deamidation is unique to O 1 atom of Asp195 forms a hydrogen bond with the N 2 atom of these effectors and is most likely an example of divergent evo- NEDD8:Gln40. This interaction, along with the backbone amides lution from a common ancestor. However, what are the key of Cys117 and Leu196 and the backbone carbonyl of Gly172, features of Cif that deliver this unique adapted activity? These forms a pattern of hydrogen bond donor/acceptor residues that ε δ can be explored by considering the two primary enzyme/substrate ensures the N 2 atom, rather than the O 1 atom, of Gln40 is interfaces formed by the Cif head and tail domains in the CifYp/ oriented toward His173 (Fig. 3F). This role is consistent with the NEDD8 and CifPl/NEDD8 complexes and their roles in pro- relatively minor impact of the CifYp(Asp195Asn) mutation on ductive substrate binding. catalysis in vitro. In contrast, the equivalent mutation in CifEc

E1834 | www.pnas.org/cgi/doi/10.1073/pnas.1112107109 Crow et al. Downloaded by guest on September 24, 2021 PNAS PLUS

Fig. 4. Comparison of the overall structure of CifPl and NEDD8 in their uncomplexed states and in the CifPl/NEDD8 complex. The structures of CifPl and 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.

(Asp187Asn) did not trigger G2/M cell-cycle arrest in infected HeLa cells (12), suggesting this subtle mutation may have functional relevance in the context of a cellular environment. The tail domains of Cifs bear no resemblance to the structurally equivalent region in cysteine proteases and appear to be an adaptation evolved to interact with NEDD8/ubiquitin substrates. Mutation of a Leu106 in CifYp (a residue that directly contacts NEDD8) to a Glu severely impairs formation of the CifYp(Cys117Ala)/NEDD8 complex, catalysis in vitro, and activity in vivo. The CifYp(Val104Glu) mutation, in a residue that does not contact NEDD8, still binds NEDD8 to WT levels. At the base of the tail domain, Asp66 (CifYp) forms hydrogen- bonding interactions with both Thr7 and Thr9 of NEDD8; the adjacent Asp67 residue is oriented away from the substrate. Neither the CifYp(Asp66Ala) nor CifYp(Asp67Ala) mutation ﬁ revealed any signi cant effect on NEDD8 binding in vitro. Fig. 5. CifYp and NEDD8 binding monitored using ITC. (A) Example of

fi BIOCHEMISTRY Binding of NEDD8 to CifYp(Asp66Arg) was still observed but binding isotherm and associated t for the CifYp(Cys117Ala)/NEDD8 in- was significantly lower than WT. Inspection of the CifYp/NEDD8 teraction. (B) Bar chart representation of the dissociation constant between fi structure suggests an Arg side chain could be accommodated at CifYp(Cys117Ala)/NEDD8, and additional variants as labeled, derived from ts to the ITC curves (Table S6). this position in the complex. A double mutant of CifEc (Asp58Ala/Asp59Ala) lost the ability to deamidate NEDD8, as assayed by nPAGE (9). Because structural data for this region of NEDD8 over ubiquitin, similar to that seen for CifBp (9). CifEc are not currently available, the potential impact of this Modeling ubiquitin on the structure of NEDD8 in the CifYp/ double mutation on the structure and how this might affect NEDD8 complex revealed only three positions at the interface function (and/or enzyme stability) are difficult to assess. that are different between these proteins: 31 (NEDD8:Glu/ No single alanine substitution variant we made in Cif sig- Yp ubquitin:Gln), 39 (Gln/Asp), and 72 (Ala/Arg). The residue at nificantly affected binding to NEDD8 in vitro, suggesting that position 72 of NEDD8/ubiquitin is known to be a key de- none of the interactions probed are, in their own right, critical to terminant of specificity in both deneddylation (38) and E1- binding. However, the introduction of bulky/charged sub- fi stitutions in key interface amino acids all resulted in disruption speci city (39) pathways. However, as supported by the structure and activity work presented here, all these changes can be ac- of the interaction. In the case of CifYp(Leu106Glu), we also show that this mutation affects catalysis in vitro and activity in vivo, commodated in the CifYp/NEDD8 complex. Similarly, docking and predict that the other bulky/charged substitutions will result the structure of CifBp on the CifYp/NEDD8 complex does not in the same effects, although this remains to be tested. Given suggest any strong selective pressure for one or another sub- these observations, as well as the extensive buried surface formed strate. Unlike CifYp and CifBp, CifEc is reported to have an between Cif and NEDD8, we conclude that shape complemen- ∼1,000-fold preference for NEDD8 over ubiquitin (9). One hy- tarity is a key feature governing the interaction between pothesis that could explain this differential activity is the lack of these proteins. conservation in amino acids of Cif (CifYp:Ile259, CifBp:His234, In this study, we have mainly focused on the Cif/NEDD8 in- CifEc:Gln251, and CifPl:Lys269; Fig. 1) that contributes to the teraction. However, Cifs can also catalyze the deamidation of recognition of the residue preceding Gln40 in the substrate Gln40 in ubiquitin. In vitro, we observe a preference of CifYp for (Gln39 in NEDD8 and Asp39 in ubiquitin). Second, CifEc has

Crow et al. PNAS | Published online June 12, 2012 | E1835 Downloaded by guest on September 24, 2021 Fig. 7. Effects of CifYp and variants on the cell-cycle arrest marker p21. (A) Fig. 6. Substrate deamidation by CifYp (and selected variants) monitored p21 accumulates in the presence of WT (wt) CifEc and CifYp but not in the using nPAGE. (A) nPAGE analysis of NEDD8 deamidation by CifYp. C/A, active site Cys mutants (C/A) or the CifYp(Leu106Glu) mutant (L/E). p21, actin, CifYp(Cys117Ala); D/N, CifYp(Asp195Asn); L/E, CifYp(Leu106Glu). (B) nPAGE and His-tagged proteins were probed with appropriate antibodies. (B) analysis of ubiquitin deamidation by CifYp.(C) Quantiﬁcation of CifYp dea- Chemiluminescence signals of p21 and actin shown in A from six [or 3 for midase activity against NEDD8 and ubiquitin from enzyme-titration experi- CifYp(Leu106Glu)] independent experiments were quantiﬁed. The averages ments (Fig. S5). CifYp (□), CifYp(Asp195Asn) (♢), CifYp(Leu106Glu) (△), and of p21 level are represented and expressed as ±SEM. Statistical differences CifYp(Cys117Ala) (○), all with a solid line and with NEDD8 as the substrate, between experimental groups were determined using one-way ANOVA with are shown. CifYp with ubiquitin (■), with a dashed line, is also shown. the Bonferroni multiple comparison posttest. **P < 0.01; ***P < 0.001.

a Ser residue at the position of an otherwise conserved Val Bank (PDB) ID code 3DQV] (16). Importantly, however, Gln40 (Val116 in Cif ), which is implicated in the reorientation of the Yp is positioned adjacent to the thioester link between the C ter- C-terminal β-strand of NEDD8, although how this could con- minus of NEDD8 and Lys724 of Cul5ctd (separated by only tribute to substrate selectivity is not clear. Although acknowl- 6.1 Å). Therefore, it is more likely that Cif-mediated deamida- edging that the published structure of Cif does not include the Ec tion of Gln40 directly interferes with CSN binding to neddylated tail domain, these are two differences that may contribute to fi ’ CRLs, although this remains to be veri ed experimentally. Fi- CifEc s NEDD8 selectivity. nally, what is the benefit to the pathogen of interfering with CRL Despite recent advances in defining the activity of Cifs, many “ ” activity? Perhaps the resulting inhibition of the cell cycle delays questions remain. First, does Cif deamidate free NEDD8 or epithelial cell renewal at the site of infection, favoring coloni- NEDD8 already conjugated to CRLs? An overlay of NEDD8 in ctd zation, or reduced CRL activity augments the activity of other the Cif/NEDD8 complexes with neddylated Cul5 -Rbx1 (16) codelivered effectors, enhancing the virulence of the pathogen. shows it is unlikely that Cif can interact with neddylated cullins in By targeting CRLs, Cifs could interfere with the stability of fi a catalytically competent orientation (because of a signi cant hundreds of substrates, suggesting that this effector may modu- clash), suggesting that Cifs deamidate NEDD8 before conjuga- late many critical host cell functions, including cell proliferation, tion. Further, when CifBp is overexpressed in HEK293T cells apoptosis, differentiation, and immune responses (7). with Cullin1, only very weak interaction is observed (22). Second, Cifs are part of a growing number of microbial products that what is the precise mechanism by which NEDD8:Gln40 deami- interfere with ubiquitination pathways in eukaryotic cells. Future dation leads to stabilization of neddylated CRLs? A recent study studies will undoubtedly use Cifs not only to address the role of concluded that Cif-mediated NEDD8 deamidation inhibits CRL these pathways in host/pathogen interactions but as tools to deneddylation by the COP9 signalosome (CSN) in vivo (22). This dissect the role of such pathways in other aspects of cell biology. study proposed two mechanisms by which this could be achieved: (i) Deamidation of Gln40 prevents NEDD8-induced conforma- Materials and Methods

tional changes in CRLs that are important for recognition by Cloning of Cifs and NEDD8 for Protein Production. The cloning of CifYp, CifBp, CSN, or (ii) deamidation of Gln40 directly inhibits recruitment CifEc, and CifPl has been described previously (10, 41). In this study, we of CSN to neddylated CRLs. The latter supports hypotheses that generated unique constructs for expression of CifBp and CifYp using existing ′ deamidated ubiquitin impairs ubiquitination pathways, because vectors as templates. For CifBp, we used primers 5 -AAGTTCTGTTTCA- GGGCCCGatgataacgccgatcatttcatcg-3′ (Forward) and 5′-ATGGTCTAGAAA- the Gln40 side chain is involved in interaction with E3 ligases (9, ′ ﬁ GCTTTAgccaaggccggcgacgtattgtgc-3 (Reverse) to amplify the region 40). It is dif cult to see how Gln40 deamidation could alter the encoding the full-length protein (23). These primers included DNA sequen- structure of neddylated CRLs; this residue is presented to bulk ces (capitalized letters) that enabled recombination-based cloning into the ctd solvent in the neddylated Cul5 -Rbx1 structure [Protein Data pOPIN-F expression vector using published procedures (42).

E1836 | www.pnas.org/cgi/doi/10.1073/pnas.1112107109 Crow et al. Downloaded by guest on September 24, 2021 PNAS PLUS To produce recombinant CifYp in a soluble form, we generated an and 0.65 μL of the crystallization reagent. Diffraction quality crystals of CifYp N-terminally truncated construct of this protein using primers 5′-AAGTTCT- (Cys117Ala)/NEDD8 crystals were grown from 2.2 M sodium malonate, 44 GTTTCAGGGCCCGgtttcacattccataaataacccttcg-3′ (Forward) and 5′-ATGGTC- mM Bis-Tris propane (pH 7), 66 mM Bis-Tris propane (pH 8) using a 1-μLsitting TAGAAAGCTTTAattacagtgagttttaatgattgacatattg-3′ (Reverse) that ampli- drop composed of 0.35 μL of the protein sample [∼500 μMcomplex,20mM ﬁed residues 33–290 of the native sequence. The resulting PCR product was Hepes (pH 7.5), 150 mM NaCl], and 0.65 μL of the crystallization reagent.

cloned into pOPIN-F as above. We also generated the CifYp(Cys117Ala) mutant in pOPIN-F using the same primers and a template DNA that already X-Ray Data Collection, Structure Solution, Reﬁnement, and Validation. Crystals

included this mutation. of CifPl(Cys123Ser)/NEDD8 and CifYp(Cys117Ala)/NEDD8 were frozen in DNA encoding full-length NEDD8 was amplified from a synthetic template a cryoprotectant solution composed of 75% of the mother liquor (taken obtained from Geneart and PCR primers 5′-AGGAGATATACCATGctaatta- directly from the crystallization plate) and 25% ethylene glycol. All X-ray aagtgaagacgctgaccggaaag-3′ (Forward) and 5′-GTGATGGTGATGTTTctgcct- data were processed with iMosflm (43) and scaled with Scala (44), as aagaccacctcctcctctcagagc-3′ (Reverse). The resulting product was cloned into implemented in the CCP4 suite (45). X-ray data collection statistics are given pOPIN-E (42), generating a C-terminal 6× His-tag. in Table 1. fi The DNA sequence of all constructs was veri ed by sequencing. The structure of CifPl(Cys123Ser)/NEDD8 was solved by molecular replacement with data collected on a Rigaku RU-H3RHB generator/Mar345

Mutagenesis of CifYp and NEDD8. All the additional mutants established in the detector and MolRep (45), using search models derived from CifPl and CifYp(Cys117Ala) background were produced by Genscript, using the pOPIN- NEDD8 [PDB ID codes 3GQY (CifPl) and 1NDD (NEDD8)]. Iterative cycles of F:CifYp(Cys117Ala) plasmid as a template. The Gln40Glu and Gln40Ala refinement with Refmac5 (45) and manual rebuilding with Coot (46) gen- mutants of NEDD8 were also produced by Genscript, using the pOPIN-E: erated a model that was fitted to high-resolution data (from the same NEDD8 plasmid as a template. crystal) obtained at beamline I02 of the Diamond Light Source (Oxford, United Kingdom). Refinement/rebuilding cycles as above generated the final

Expression and Purification of Cif Proteins. CifYp and CifBp were expressed model. Anisotropic B-factors and alternate conformations were added to- fi from pOPIN-F, and CifEc and CifPl were expressed from pET28a-based plas- ward the end of re nement. mids. Soluble CifYp was obtained using E. coli SoluBL21 (Gelantis); expression The structure of CifYp(Cys117Ala)/NEDD8 was solved by molecular re- of all other Cifs used E. coli BL21(DE3). Bacterial cultures were grown in LB placement with data collected at beamline I24 of the Diamond Light broth at 37 °C, supplemented with carbenicillin (pOPIN-F, 100 mg/L) or Source and MolRep. An edited version of the CifPl(Cys123Ser)/NEDD8 was kanamycin (pET28a, 30 mg/L), before induction with 1 mM isopropyl-β-D- used as a model (essentially polyalanine traces were generated). The thiogalactopyranoside and overnight growth at 16 °C. Cells were pelleted at resulting phases were modified by Parrot (47); Buccaneer (47) was used to 5,000 × g, frozen, and stored at −80 °C until ready for purification. Thawed rebuild the structure with the correct sequence. Iterative cycles of re- cell pellets were resuspended in 50 mM Hepes (pH 7.8), 300 mM NaCl, and finement with Refmac5 and manual rebuilding with Coot generated the 25 mM imidazole before being lysed by sonication. Unbroken cells and de- final model. bris were cleared by centrifugation at 30,000 × g for 25 min. Cleared lysates Structure validation used Coot and Molprobity (48). A selection of re- were loaded onto preequilibrated Ni2+-immobilized metal ion affinity finement and validation statistics is given in Table 1. Structure-based chromatography (IMAC) columns; washed extensively in load buffer; and overlays were calculated using secondary-structure matching algorithms step-eluted in 50 mM Hepes (pH 7.8), 300 mM NaCl, and 250 mM imidazole. as implemented in Superpose from the CCP4 suite (25, 45). The coor-

The eluate was directly loaded onto a Hi-Load 26/60 Superdex 75 gel fil- dinates and structure factors for the CifYp/NEDD8 and CifPl/NEDD8 com- tration column (GE Healthcare); equilibrated; and run in 20 mM Hepes plexes have been submitted to the PDB with ID codes 4F8C (CifYp/NEDD8) (pH 7.5), 150 mM NaCl, and 1 mM DTT (DTT was excluded from the buffer for and 4FBJ (CifPl/NEDD8). purification of proteins used in ITC experiments). Proteins were concentrated by ultrafiltration to between 350 μM and 950 μM before aliquoting Protein/Protein Interaction Studies (ITC). Cifs and NEDD8 were both prepared and flash-freezing in liquid nitrogen. All protein aliquots were stored frozen in 20 mM Hepes (pH 7.5) and 150 mM NaCl, and were diluted consistently until required for experiments. into the same buffer. All ITC experiments were performed with a MicroCal 205 calorimeter in high gain mode. In a typical experiment, the calorimetry Preparation of NEDD8 and Ubiquitin. NEDD8 was expressed from pOPIN-E cell was filled with 205 μLofCifat∼100 μM before making sequential using E. coli SoluBL21. Purification proceeded as described for Cifs, and DTT injections of ∼1,200 μM NEDD8 from the syringe up to a final cumulative was not included in the gel filtration buffer. injection volume of 38 μL. Experiments were conducted at 25 °C, with Ubiquitin was purchased from Sigma (bovine, identical sequence to human typical injections of between 1.0 μL and 2.0 μL at 180-s intervals. Two

ubiquitin) and redissolved in 20 mM Hepes (pH 7.5) and 150 mM NaCl for use. control experiments were performed to ensure that the heat of dilution of BIOCHEMISTRY NEDD8 or Cif would not be problematic under these experimental con- Purification and Crystallization of Cif/NEDD8 Complexes. To generate Cif/ ditions; these involved direct injections of NEDD8 into buffer and injections NEDD8 complexes for purification and subsequent crystallization, we of buffer into a solution of Cif. Binding isotherms were fitted to the in- adopted a “cosplitting” approach, where pelleted bacterial cells previously tegrated calorimetric data using Origin (Microcal). Each binding experiment induced to express individual Cif proteins were mixed and resuspended with was performed at least three times (except for the Cys117Ala/Asp66Arg pelleted cells containing expressed NEDD8. The mixture of resuspended cells variant, which was performed twice), with the mean and SD calculated for was then sonicated, and proteins were purified as above, retaining elution each variant. peaks that contain complexes as judged by SDS/PAGE. We attempted to saturate Cif with NEDD8 by adding excess culture expressing this second nPAGE to Monitor Enzyme Activity. A total of 700 pmol of NEDD8, NEDD8 protein. Saturation was evident from the presence of a large peak corre- (Q40A), and NEDD8(Q40E) was incubated with 0.1 pmol of CifYp (or variants) sponding to free NEDD8 in the gel filtration profile at a high retention for 30 min at 30 °C. Total reaction volume was 10 μL. The reaction mixture volume and the absence of a peak or shoulder at elution volumes that was chilled on ice, diluted with 1:1 loading buffer [50 mM glycine (pH 10.0), μ would correspond to free Cifs. For CifBp and CifYp, we also produced samples 20% (vol/vol) glycerol], and 10 L was loaded onto 12.5% nPAGE gels in which the N-terminal His-tag was removed using 3C protease (samples buffered with 50 mM glycine at pH 10.0 (gels were run with the same were concentrated to ∼700 μM and treated with 0.01 mg/mL 3C protease buffer). Therefore, 350 pmol of substrate was loaded on the gel. Proteins overnight at room temperature). were visualized with InstantBlue staining, and the bands quantified using ImageJ (National Institutes of Health). The same protocol was used for the Crystallization of Cif/NEDD8 Complexes. We produced Cif/NEDD8 complexes ubiquitin assays, except 1.0 pmol of CifYp (or variants) was incubated with

using CifYp, CifBp,CifEc, and CifPl with various catalytic site mutants. Extensive 700 pmol of substrate and gels were stained with Coomassie Blue. screening of crystallization conditions for these complexes using robotic For the titration experiments, the range of enzyme quantities used is

setups identified crystals for CifPl(Cys123Ser)/NEDD8 and CifYp(Cys117Ala)/ shown in Fig. 6C and Fig. S5. NEDD8. The latter was from a sample treated with 3C protease. CifPl (Cys123Ser)/NEDD8 crystals were confirmed to comprise both proteins using Lipofection Experiments. Lipofection assays of purified Cif proteins were

MS. Diffraction quality crystals of CifPl(Cys123Ser)/NEDD8 were obtained conducted essentially as previously described (10). HeLa cells (CCL-2; Amer- from 20% (vol/vol) PEG 4000, 200 mM sodium acetate, and 100 mM Mes (pH ican Type Culture Collection) were cultured in DMEM (Invitrogen) supple- 6.7) using a 96-well sitting drop plate and a drop composed of 0.35 μLof mented with 10% FCS (Eurobio) and 80 mg/L gentamicin at 37 °C in a 5%

protein solution [∼600 μM complex in 20 mM Hepes (pH 7.5), 150 mM NaCl] CO2 atmosphere. For lipofection assays, 80 μL of puriﬁed CifYp,CifEc (500 μg/

Crow et al. PNAS | Published online June 12, 2012 | E1837 Downloaded by guest on September 24, 2021 mL), or PBS as a negative control was added to one Bio-PORTER tube visualized with HRP-conjugated secondary antibody. Acquisitions were per- (Genlantis) and resuspended in 420 μL of DMEM. The samples were added to formed with a Molecular Imager ChemiDoc XRS system (Bio-Rad). Protein the HeLa cells in six-well plates and incubated for 4 h before being replaced levels were quantiﬁed with Quantity One software (Bio-Rad) and normal- by fresh growth medium and further incubated for 20 h. ized with the actin level. For Western blot analyses, ∼6 × 105 cells were lysed in 80 μL of SDS/PAGE sample buffer, sonicated for 2 s to shear DNA, and then boiled for 5 min. ACKNOWLEDGMENTS. We thank Dr. Jean-Philippe Nougayrède (Toulouse) – Protein samples were resolved on either 10% or 4 12% NuPage gradient and all the M.J.B. group for discussions. We acknowledge the staff of gels (Invitrogen) and blotted on PVDF membranes. Membranes were the Diamond Light Source synchrotron radiation source for access to blocked in 10 mM Tris (pH 7.8), 150 mM NaCl, 0.1% Tween 20, and 5% data collection facilities. This work was supported by Grant F008732 of the −1 nonfat dry milk, and then probed with primary antibody (0.5 mg/mL )in Biotechnology and Biological Sciences Research Council, United Kingdom (to the same buffer. Primary antibodies used were anti-actin (ICN), anti-p21 (Cell M.J.B.), a Royal Society (United Kingdom) University Research Fellowship (to Signaling Technology), and anti-histidine (Qiagen). Bound antibodies were M.J.B.), and the Ligue Nationale Contre le Cancer (F.T.).

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