Structure of the Chlamydia Protein CADD Reveals a Redox Enzyme That Modulates Host Cell Apoptosis
The Chlamydia protein CADD (Chlamydia protein associating with death domains) has been implicated in the modulation of host cell apoptosis via binding to the death domains of tumor necrosis factor family receptors. Transfection of CADD into mammalian cells induces apoptosis. Here we present the CADD crystal structure, which reveals a dimer of seven-helix bundles. Each bundle contains a di-iron center adjacent to an internal cavity, forming an active site similar to that of methane mono-oxygenase hydrolase. We further show that CADD mutants lacking critical metal-coordinating residues are substantially less effective in inducing apoptosis but retain their ability to bind to death domains. We conclude that CADD is a novel redox protein toxin unique to Chlamydia species and propose that both its redox activity and death domain binding ability are required for its biological activity. The Chlamydia protein CADD (Chlamydia protein associating with death domains) has been implicated in the modulation of host cell apoptosis via binding to the death domains of tumor necrosis factor family receptors. Transfection of CADD into mammalian cells induces apoptosis. Here we present the CADD crystal structure, which reveals a dimer of seven-helix bundles. Each bundle contains a di-iron center adjacent to an internal cavity, forming an active site similar to that of methane mono-oxygenase hydrolase. We further show that CADD mutants lacking critical metal-coordinating residues are substantially less effective in inducing apoptosis but retain their ability to bind to death domains. We conclude that CADD is a novel redox protein toxin unique to Chlamydia species and propose that both its redox activity and death domain binding ability are required for its biological activity. Chlamydiae are obligate intracellular bacteria and the causative agents of important sexually transmitted and disabling ocular (blinding trachoma) human diseases (1Antilla T. J. Am. Med. Assoc. 2001; 285Google Scholar). Chlamydia engages in a unique relationship with its host. Upon entering host cells, the parasite starts a biphasic developmental cycle from the infectious form, called an elementary body, to a non-infectious, vegetative growth form, called a reticulate body, and then eventually back to the replication-incompetent infectious form (2Schachter J. Curr. Top. Microbiol. Immunol. 1988; 138: 109-139PubMed Google Scholar). After the transition back to the infectious form, the host cell dies and releases its infectious load (3Belland R.J. Scidmore M.A. Crane D.D. Hogan D.M. Whitmire W. McClarty G. Caldwell H.D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13984-13989Crossref PubMed Scopus (159) Google Scholar). To accommodate its life cycle, Chlamydia may inhibit apoptosis during the early stages of infection (4Fischer S.F. Schwarz C. Vier J. Hacker G. Infect. Immun. 2001; 69: 7121-7129Crossref PubMed Scopus (106) Google Scholar, 5Geng Y. Shane R.B. Berencsi K. Gonczol E. Zaki M.H. Margolis D.J. Trinchieri G. Rook A.H. J. Immunol. 2000; 164: 5522-5529Crossref PubMed Scopus (118) Google Scholar) and promote apoptosis at later stages (6Schoier J. Ollinger K. Kvarnstrom M. Soderlund G. Kihlstrom E. Microb. Pathog. 2001; 31: 173-184Crossref PubMed Scopus (31) Google Scholar, 7Perfettini J.L. Hospital V. Stahl L. Jungas T. Verbeke P. Ojcius D.M. Biochimie (Paris). 2003; 85: 763-769Crossref PubMed Scopus (31) Google Scholar). Recently, the Chlamydia protein CADD 1The abbreviations used are: CADD, Chlamydia protein associating with death domains; MAD, multiwavelength anomalous diffraction; GFP, green fluorescent protein; EGFP, enhanced GFP; GST, glutathione S-transferase; r.m.s.d., root mean square deviation; MMOH, methane monooxygenase; ICP-AAS, inductively coupled atomic absorption spectroscopy; PQQ, pyrrolo-quinoline-quinone; wt, wild type. has been shown to associate with tumor necrosis factor family proteins and to induce apoptosis when transfected into a variety of mammalian cell lines (8Stenner-Liewen F. Liewen H. Zapata J.M. Pawlowski K. Godzik A. Reed J.C. J. Biol. Chem. 2002; 277: 9633-9636Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). CADD has no close homologues but does show 18% sequence identity with coenzyme PQQ (pyrrolo-quinolinequinone) synthesis protein C (PqqC) family members, which are part of the six-step PQQ synthesis pathway in bacteria (9Meulenberg J.J. Sellink E. Loenen W.A. Riegman N.H. van Kleef M. Postma P.W. FEMS Microbiol. Lett. 1990; 59: 337-343Crossref PubMed Google Scholar). However, homologues of other members of the pathway are not found in Chlamydia species for which genome information is available. Indeed, ectopic expression of PqqC from Klebsiella pneumoniae failed to cause apoptosis, demonstrating the specificity of CADD-induced cell death (8Stenner-Liewen F. Liewen H. Zapata J.M. Pawlowski K. Godzik A. Reed J.C. J. Biol. Chem. 2002; 277: 9633-9636Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). CADD is expressed late in the infectious cycle of Chlamydia trachomatis and is secreted into the host cytoplasm, where it co-localizes with tumor necrosis factor receptors in the proximity of the inclusion body. Sequence comparisons had suggested that CADD contains a death domain. Here we present the crystal structure of CADD, which reveals an iron-containing redox enzyme that bears no resemblance to death domains. Mutagenesis of the active site of CADD reduced but did not eliminate its apoptotic activity, suggesting that both its catalytic activity and death domain binding activities contribute to its biological activity. Mutation, Expression, and Purification of CADD—The open reading frame encoding CADD, CT610 (GI: 3329055) from C. trachomatis was subcloned into pcDNA3-hemagglutinin (Invitrogen), pGEX-4T (Amersham Biosciences), pet21d (Invitrogen), and PEGFP-C2. The following mutations, Y170F (CADD-mut1) and E81A/H88A/Y170F/H174A (CADD-mut2) were introduced using the QuikChange kit (Stratagene), confirmed by DNA sequencing, subcloned into pet21d (Invitrogen), pGEX-4T, PEGFP-C2 and pDS-RED-C2, and transformed into Escherichia coli XLBlue. Glutathione S-transferase (GST) fusion proteins were obtained by induction with 0.1 mm isopropyl-β-d-thiogalactopyranoside at 25 °C for 8 h and then purified using glutathione-Sepharose (Amersham Biosciences). After thrombin cleavage, CADD was further purified on an s200 gel filtration column (Aekta-FPLC, Amersham Biosciences), concentrated to 12 mg/ml (AMICON), and flash-frozen in liquid nitrogen for long term storage at –80 °C. The selenomethionine-substituted protein was expressed as described (10Harrison C.J. Bohm A.A. Nelson H.C. Science. 1994; 263: 224-227Crossref PubMed Scopus (220) Google Scholar) and purified as for the wild type, except that 5 mm tris(2-carboxyethyl)phosphine was added to the dialysis and gel filtration buffers. Crystallization—Purified CADD was crystallized by the vapor diffusion method at room temperature using a sparse matrix screen (Hampton). Sitting and hanging drops consisting of 3 μl of precipitant solution (10% (v/v) polyethylene glycol 12000, 20 mm cacodylate, pH 6.5) and 3 μl of protein solution (12 mg/ml protein) yielded crystals within 3–5 days. Crystals grew as very thin plates with dimensions of 200 × 200 × 20 μm in space group C2221. The crystal structure was determined by a selenium MAD experiment using a seleno-methionine substituted protein (10Harrison C.J. Bohm A.A. Nelson H.C. Science. 1994; 263: 224-227Crossref PubMed Scopus (220) Google Scholar). For data collection, crystals were transferred into cryobuffer (crystallization buffer with 25% (v/v) glycerol) and flash-cooled in liquid nitrogen. Data Collection, Structure Solution, and Refinement—The three-wavelength MAD data set was collected from one single crystal, using synchrotron radiation at beamline X12B of the National Synchotron Light Source. Oscillation data were recorded in frames of 1° through a continuous angular range of 120° for the peak (λ = 0.9791 Å), the high energy remote (λ = 0.925 Å), and the inflection point (λ = 0.9794 Å). The native data set was collected at beamline X9B of National Synchotron Light Source. All data were processed with the programs DENZO and SCALEPACK (11Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38570) Google Scholar). The CADD structure was phased and traced using the program SOLVE/RESOLVE (12Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 849-861Crossref PubMed Scopus (3220) Google Scholar). Model building and refinement were carried out in O (13Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13011) Google Scholar) and REFMAC5 (14P4 CC Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19768) Google Scholar). The final CADD model comprises three protein monomers (residues A7–A219, B7–B219, C7–C219), 6 Fe2+ ions with 3 closely bound putative water molecules, and 176 water molecules. Residues 1–6 and 220–231 were not visible in the electron density maps and therefore were not included in the model. Statistics for data collection, refinement, and model quality are summarized in Table I. Surface calculations were carried out with the CASTP server (15Liang J. Edelsbrunner H. Woodward C. Protein Sci. 1998; 7: 1884-1897Crossref PubMed Scopus (868) Google Scholar) and the protein-protein-interaction server (16Jones S. Thornton J.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13-20Crossref PubMed Scopus (2275) Google Scholar). Figures were drawn with SPOCK (17Christopher J.A. SPOCK. The Center for Macromolecular Design, Texas A&M University, College Station, TX1998Google Scholar) and PYMOL (DeLano Scientific LLC).Table ICrystallographic statistics of CADDNativeSe-λ1Se-λ2Se-λ3Data collectionSpace groupC2221C2221C2221C2221Cell dimensions (Å)a77.5577.6377.6277.51b192.97193.33193.38193.70c93.7493.9993.9794.11National Synchotron Light Source beamlineX9BX12BX12BX12BWavelength (Å)0.9540.97930.97850.9611Resolution (Å)95-2.530-3.130-3.130-3.1Reflections (observed)98,53295,73488,84793,278Reflections (unique)24,06913,11713,07913,212Completeness (%)96.8 (94.2)99.8 (100.0)99.7 (100.0)99.7 (99.8)I/σ(I)11.2 (2.3)7.1(1.4)7.4 (1.5)6.3 (1.2)RmergeaRmerge = Σ I — [I] /ΣI, where I is the observed intensity and [I] is the average intensity from multiple observations of symmetry-related reflections, values in parentheses correspond to the highest resolution shell. (%)6.1 (40.1)15.1 (49.3)15.7 (53.6)18.1 (62.3)Phasing (MAD)Resolution range30-3.5Number of selenium sites12Figure of merit0.44RefinementResolution range (Å)95-2.5RcrystbRcryst = Σ|Fo — Fc|/Σ|Fo|. (%)21.66RfreecRfree = same as Rcryst but comprises a test set (5% of total reflections), which was not used in model refinement. (%)25.85Protein atoms5,189Iron atoms6Solvent molecules176r.m.s. deviationsBond angles (°)1.51Bond lengths (Å)0.015a Rmerge = Σ I — [I] /ΣI, where I is the observed intensity and [I] is the average intensity from multiple observations of symmetry-related reflections, values in parentheses correspond to the highest resolution shell.b Rcryst = Σ|Fo — Fc|/Σ|Fo|.c Rfree = same as Rcryst but comprises a test set (5% of total reflections), which was not used in model refinement. Open table in a new tab Cell Culture, Transfections, and Apoptosis Measurements—HeLa cells were maintained in Dulbecco's modified Eagle's medium (Irvine Scientific) and supplemented with 10% fetal bovine serum, 1 mm l-glutamine, and antibiotics. Cells (106) were transfected with PEGFP-C2 plasmids containing CADDwt, CADD-mut1, and CADD-mut2, using LipofectAMINE (Invitrogen) following the vendor's protocol. Both floating and adherent cells were recovered 1 day later and pooled, and the percentage of transfected (green fluorescent) cells with nuclear apoptotic morphology was determined by staining with 0.1 μg/ml 4′,6-diamidino-2-phenylindole (mean ± S.D.; n = 3). Cytosolic extracts from HeLa cells were subjected to immunoblotting and probed with rabbit polyclonal anti-green fluorescent protein (GFP) antibody (Invitrogen) for the presence of GFP-CADD fusion proteins. Protein Binding Assays—A plasmid containing DR5 was in vitro transcribed and translated in the presence of l-[35S]methionine using the TnT kit from Promega. GST-CADD, GST-CADD-mut1(data not shown), GST-CADD-mut2, and control GST-CD40 (cytosolic domain) fusion proteins were immobilized on glutathione-Sepharose at 1 μg/μl and incubated with in vitro translated target proteins for 2 h at 4 °C. Beads were then washed four times in 1 ml of 140 mm KCl, 20 mm Hepes, pH 7.5, 5 mm MgCl2, 2 mm EGTA, 0.5% Nonidet P-40, and analyzed by SDS-PAGE/fluorography. Mass Spectrometry and ICP-AAS—Matrix-assisted laser desorption/ionization-time of flight, peptide mapping, and ICP-AAS-spectrometric analysis on the purified CADD protein were accomplished using standard techniques at the Facility for Mass Spectrometry at the Scripps Research Institute in La Jolla. Coordinates—Coordinates and structure factors for CADD have been deposited with the Protein Data Bank (www.rcsb.org/pdb) under accession code 1RCW. CADD Structure—Recombinant CADD from C. trachomatis was expressed in E. coli, purified, and crystallized. The crystal structure was determined by a selenium MAD experiment (10Harrison C.J. Bohm A.A. Nelson H.C. Science. 1994; 263: 224-227Crossref PubMed Scopus (220) Google Scholar). CADD is a 231-residue protein, molecular mass = 26,734 Da, which forms a homo-dimer in solution, as judged by gel filtration. The CADD monomer is cylindrical with approximate dimensions of 45 × 29 × 37 Å. CADD folds into a seven-helix mostly parallel/anti-parallel bundle, where six α-helices (H1, H2, H3, H4, H5, H7) partly embrace the seventh helix (H6) (see Fig. 2A). According to the Structural Classification of Proteins Data Base (18Murzin A.G. Brenner S.E. Hubbard T. Chothia C. J. Mol. Biol. 1995; 247: 536-540Crossref PubMed Scopus (5594) Google Scholar), CADD belongs to the “heme-oxygenase” fold. Helices H1, H3, H4 are kinked and can therefore be represented as separate shorter α-helices denoted as A and B. This is especially true for helix H3, where a hairpin loop, residues 82–87, is inserted (Figs. 1A and 2).Fig. 1The overall structure of CADD. A, CADD depicted in ribbon representation, rainbow color-coded from N terminus (blue) to C terminus (red), with helices H1–H7, the two iron ions, and loop L3 labeled. B, the CADD dimer is shown normal and parallel to its long axis.View Large Image Figure ViewerDownload (PPT) The CADD dimer is formed through an interaction via helices H2 and H3A, residues 59–85 (Figs. 1B and 2). The interface-accessible surface area is 915 Å2/monomer, which accounts for 9.2% of the accessible surface area of the CADD dimer. The interaction is predominantly but a of and The similar found using the server L. C. J. Mol. Biol. PubMed Scopus Google Scholar) are: PqqC H. M. A. Reed J.C. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), with an of for the of and 18% sequence human D.J. A. Biol. 1999; PubMed Scopus Google Scholar), with an of for and sequence the of M. A. H. 1998; PubMed Scopus Google Scholar), with an of for and sequence and the of methane H. P. 1997; PubMed Scopus (159) Google Scholar), with an of for residues and sequence of the active are of to be a redox suggesting that is for of According to sequence with the server and L. W. Godzik A. Protein Sci. 2002; PubMed Scopus Google Scholar), CADD sequence with A factors S. J. 1991; PubMed Google Scholar) and can be used as a to for proteins. The helices of CADD the for a internal with a center (Figs. 2 and The electron density the presence of two ions by 6 residues (Figs. 2 and The site is in the center of the adjacent to the cavity, which as the active absorption using the presence of iron and but of in the This the presence of a di-iron judged by maps and is not in the CADD The of be to and of iron for which has been observed in crystal of proteins J. Chem. 2003; PubMed Scopus Google Scholar). The center to be and by a and a water is by two and the as as the putative which it with is by two and the water All 6 active site residues the ions are CADD proteins from Chlamydia species 2). The water is by both iron at a of (Figs. and is adjacent to and and the internal active site The density peak in for the water is by the iron and therefore not The electron density and are with a species bound to the di-iron The to the di-iron site an overall and 5 × × with a of (15Liang J. Edelsbrunner H. Woodward C. Protein Sci. 1998; 7: 1884-1897Crossref PubMed Scopus (868) Google Scholar). The is with residues and cavity, the is and contains a A of the of the with two to loop L3 and helices and the surface of the protein helices H2, H3, and the unique loop L3 (Figs. 1A and is by residues and from the di-iron site through a into a by residues and and from to the surface to residues and and The of the active site the to as through by of a of CADD and A, of CADD and the of Data Bank accession code in ribbon The ions in the active site are shown as and B, same as A a of the two di-iron The Fe2+ ions are shown as with the residues in in in of the active site in surface from CADD and The site is shown for Large Image Figure ViewerDownload (PPT) The active site of CADD is similar to that found in from E. coli Data Bank accession code The helices forming the that contains the active site can be with an of Å. The of is to a on an adjacent with the of its di-iron The is transferred to the which the of M. A. H. 1998; PubMed Scopus Google Scholar). CADD contains a to the di-iron the of the of CADD is the of for However, no are for and which are critical residues for the pathway in to J. Chem. 2003; PubMed Scopus Google Scholar). that CADD as a but a for of test and the site are in the of CADD, we two active site mutants by and their apoptotic activity through in mammalian The of was with a Y170F (CADD-mut1) To the of a we the of the of plasmid DNA were transfected into HeLa cells, a in of when with the in apoptotic activity that both CADD mutants are expressed at similar to This that the especially CADD-mut2, are by the transfected mammalian cells the To the of the active site proteins bind to death we carried out an in vitro DR5 binding not shown), and CADD wild and active site mutants show binding to death that the active site not the DR5 binding activity of CADD. The crystal structure that CADD to and PqqC The sequence and for CADD proteins are by the same but the active are not and the two proteins are therefore and H. M. A. Reed J.C. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). CADD is an unique to Chlamydia which further its as a toxin that in intracellular with the M. A. H. 1998; PubMed Scopus Google Scholar) and H. P. 1997; PubMed Scopus (159) Google Scholar), reveals di-iron active in a similar the three proteins to the and to the the helices forming the containing the active site can be with an Å. The active site of CADD is similar to that in but does not the residues of the CADD can therefore not as an but it is to that CADD, may and a on to redox However, with an Y170F show a in activity, that is not for CADD The of CADD contains and it is that one may for the of A with the di-iron center in from Data Bank accession code H. P. 1997; PubMed Scopus (159) Google Scholar) reveals of the except for a in the of in CADD, where is on the other of with A and A analysis of the active further reveals that in to and CADD an internal to the center MMOH, the as the site of where and the di-iron center through the from the of the CADD contains a similar when the to and H5, is used the other the to the loop L3 is a for a that open the to the for the of and CADD is an enzyme similar to H. P. 1997; PubMed Scopus (159) Google Scholar), which an internal active site with a di-iron center to redox on are to the by CADD. Transfection with a CADD lacking critical metal-coordinating residues a the di-iron site and the apoptotic activity of CADD. at the active which is within the not interaction with death which that the induction of apoptosis by CADD both the of death receptors as as its redox activity. We for and