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

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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, re- lated. 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 stabiliza- tion 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 re- newal 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-
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