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Crystal structure of virulence factor CJ0248 from Campylobacter jejuni at 2.25 Å resolution reveals a new fold

Year: 2005

Type: article

Abstract: In order to extend the structural coverage of eukaryotic proteins in the Protein Family database (PFAM),1 we selected 331 bacterial homologues of proteins represented in the mouse genome from the American Type Culture Collection (ATCC) proteome database (http://www.ebi.ac.uk/proteome/ProteomeSource.html). One of these, corresponding to the CJ0248 gene of Campylobacter jejuni, belongs to a large protein family that includes members such as hydrolases and signal-transduction proteins.2 This family is highly conserved, with hundreds of homologs in all kingdoms of life, and also includes metal-dependent phosphohydrolases (PF01996). CJ0248 encodes a protein with a molecular weight of 31,828 Da (residues 1–285) and a calculated isoelectric point of 5.84. CJ0248 is a virulence factor of yet unknown function that has been identified along with 22 other genes of Campylobacter jejuni to be involved in colonization of the chick gastrointestinal tract.3 Strains deficient in CJ0248 are significantly attenuated. Campylobacter jejuni is the leading cause of bacterial gastroenteritis in humans in developed countries throughout the world. This bacterium frequently promotes a commensal lifestyle in the gastrointestinal tracts of many animals, including birds, and consumption or handling of poultry meats is a prevalent source of C. jejuni infection in humans.3 Here, we report the crystal structure of CJ0248, which was determined using the semi-automated, high-throughput pipeline of the Joint Center for Structural Genomics (JCSG).4 Virulence factor CJ0248 from Campylobacter jejuni (gi: 6967725, Swiss-Prot: Q9PIP7) was amplified by polymerase chain reaction (PCR) from genomic DNA using PfuTurbo (Stratagene) and primer pairs encoding the predicted 5′- and 3′-ends. The PCR product was cloned into plasmid pMH4, which encodes an expression and purification tag (MGSDKIHHHHHH) at the amino terminus of the full-length protein. The cloning junctions were confirmed by sequencing. Protein expression was performed in a selenomethionine-containing medium using the Escherichia coli methionine auxotrophic strain DL41. Lysozyme was added to the culture at the end of fermentation to a final concentration of 250 μg/mL. Bacteria were lysed by sonication after a freeze/thaw procedure in Lysis Buffer [50 mM Tris pH 8.0, 50 mM NaCl, 10 mM imidazole, 1 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP)], and the cell debris was pelleted by centrifugation at 32,500 × g for 30 min. The soluble fraction was applied to a nickel-chelating resin (GE Healthcare) pre-equilibrated with Lysis Buffer. The resin was washed with Wash Buffer [50 mM Tris pH 8.0, 300 mM NaCl, 40 mM imidazole, 10% (v/v) glycerol, 1 mM TCEP], and the target protein was eluted with Elution Buffer [20 mM Tris pH 8.0, 300 mM imidazole, 10% (v/v) glycerol, 1 mM TCEP]. The eluate was diluted 10-fold with Buffer Q [20 mM Tris, pH 7.9, 50 mM NaCl, 5% (v/v) glycerol, 0.25 mM TCEP] and applied to a RESOURCE Q column (GE Healthcare) pre-equilibrated with the same buffer. The target protein eluted in the flow-through fraction was buffer-exchanged into Crystallization Buffer [20 mM Tris, pH 7.9, 150 mM NaCl, 0.25 mM TCEP] and concentrated for crystallization assays to 15 mg/mL by centrifugal ultrafiltration (Millipore). The molecular weight and oligomeric state of the target protein were determined using a 1.0 × 30 cm Superdex 200 column (GE Healthcare) in combination with dynamic light scattering (Wyatt Technology). The mobile phase consisted of 20 mM Tris pH 8.0, 150 mM NaCl. The protein was crystallized using the nanodroplet vapor diffusion method5 with standard JCSG crystallization protocols.4 The crystallization reagent contained 20% (w/v) polyethylene glycol (PEG)-6000, 1.0 M LiCl, 0.1 M HEPES pH 7.5. Ten percent (v/v) ethylene glycol (final concentration) was included as a cryoprotectant. The crystals were indexed in the monoclinic space group C2 (Table I). Single-wavelength anomalous diffraction data were collected at the Advanced Light Source (ALS, Berkeley, USA) on beamline 5.0.2 at wavelengths corresponding to the inflection (λ1) of a selenium single-wavelength anomalous dispersion (SAD) experiment. The datasets were collected at 100K using an ADSC CCD detector. The SAD data were integrated and reduced using Mosflm8 and then scaled with the program SCALA from the CCP4 suite.6 Data statistics are summarized in Table I. The structure was determined with 2.25 Å selenium SAD data using the CCP4 suite6 and SOLVE/RESOLVE.9 Automatic model building was performed with iterative runs of RESOLVE.9 Model completion and refinement were performed using O10 and REFMAC5.6 Refinement statistics are summarized in Table I. The final model includes four monomers (A–D) and 193 water molecules in the asymmetric unit. No electron density was observed for residues 34 and 35 in chain A, residues 1–4, 33–36, 94–97, and 282–285 in chain B, residues 282–285 in chain C, residues 1–4, 31–36, 94–97, 282–285 in chain D, and of the expression and purification tag in all chains, except for one Histidine in chain A. Analysis of the stereochemical quality of the model was accomplished using the AutoDepInputTool (http://deposit.pdb.org/adit/), MolProbity,11 SFcheck 4.0,6 and WHAT IF 5.0.12 Figures were prepared with PYMOL (DeLano Scientific). Atomic coordinates and experimental structure factors for CJ0248 from Campylobacter jejuni have been deposited in the PDB and are accessible under the code 1vqr. The crystal structure of CJ0248 [Fig. 1(a)] was determined to 2.25 Å resolution using the SAD method. Data collection, model, and refinement statistics are summarized in Table I. The final model includes four monomers (A–D), for which chain A (residues 1–33 and 36–285) is the most complete, and 193 water molecules in the asymmetric unit. The Matthews' coefficient (Vm)13 for CJ0248 is 2.58 Å3/Da, and the estimated solvent content is 51.9%. The Ramachandran plot, produced by MolProbity,11 shows that 98.4% and 1.6% of the residues are in favored and allowed regions, respectively. Crystal structure of CJ0248 from Campylobacter jejuni. (A) Ribbon diagram of CJ0248 color-coded from N-terminus (blue) to C-terminus (red) showing the domain organization. Helices H1–H19 are indicated. (B) Diagram showing the secondary structure elements in CJ0248 superimposed on its primary sequence. The α-helices, β-bulges, and disordered regions (dots) are indicated for chain A. CJ0248 is a compact, helical molecule with approximate dimensions of 60 × 55 × 40 Å3. CJ0248 is composed of sixteen α-helices (H1–H4, H6, H7, H9–H14, H16–H19) and three 310-helices (H5, H8, H15) [Fig. 1(a,b)]. The total α-helical and 310-helical content is 65.5% and 3.2%, respectively. CJ0248 folds into a complex arrangement of helices wrapped around a 40 Å long, highly-kinked, hydrophobic helix H10 in the center of the molecule. The structure reveals a 6 Å wide and 15 Å deep pocket adjacent to the linker between helices H11/H12 and H16/H17. The pocket is lined by several, conserved, charged, residues (Arg147, Arg213, and Asp233), with a negatively-charged entrance formed by the conserved Asp233 as well as Asp240, Gly241, and Asn246 [Fig. 2(a)]. The location of this pocket and its sequence conservation points to its possible role as the active site of this protein. The crystallographic packing of the CJ0248 structure indicates that a monomer is the biologically relevant form. The monomer is also consistent with results from analytical size exclusion chromatography in combination with static light scattering. (A) CJ0248 in surface representation colored according to electrostatic potential in a range where red is negative (−75 kT/e) and blue is positive (+75 kT/e). Note the deep pocket guarded by a negatively charged entrance. (B) Superposition of CJ0248 (blue ribbon; same view as A) with the catalytic core domain (residues 230–345 and 377–393) of human phosphodiesterase 4B2B (PDB: 1f0j; yellow ribbon with zinc depicted as yellow sphere). (C) Same view as B, but zoomed into the active site of human phosphodiesterase with zinc (purple sphere) and coordinating residues shown in white sticks. CJ0248 residues in the postulated active site are shown in blue sticks and labeled in bold. The HD motif (His274 and Asp275 versus Leu146 and Arg147) and zinc center are not conserved in CJ0248. A structural similarity search, performed with the coordinates of CJ0248 using the DALI server,14 revealed no structural similarity to full-length CJ0248, indicating that this is a new fold. Interestingly, CJ0248 shows only distant structural similarity to members of its putative protein family (PF01996). The closest structural homolog is the catalytic domain of human phosphodiesterase 4B2B [Protein Data Bank (PDB): 1f0j].15 The structural alignment covers residues 110–240 of CJ0248 and shows a root mean square deviation (RMSD) of 4.2 Å with 7% sequence identity [Fig. 2(b)]. The superposition reveals that the active site of human phosphodiesterase, which contains a Zn2+ coordinated by conserved histidine (H) and aspartate (D) residues including the conserved HD motif (274-HD-275), superimposes well with the deep pocket in CJ0248. However, the CJ0248 structure lacks three of the metal coordinating residues, does not contain a metal site and, therefore, cannot function as a metal-dependent phosphohydrolase. In CJ0248, the conserved HD-sequence motif is replaced by 146-LR-147, located in the kink of helix H11 [Fig. 2(c)].2 The side chain of Arg147 replaces the zinc atom and interacts with the side chain of the conserved Asp233, which in turn hydrogen bonds with Arg213 to form an Arg-Asp-Arg triad. These findings indicate that CJ0248 is not a metal-dependent phosphohydrolase and is an unusual member of the protein family PF01996. Instead, CJ0248 likely represents a new family of proteins with more than 200 sequence homologs in archaea, bacteria, and eukaryotes. Models for CJ0248 homologs can be accessed at http://www1.jcsg.org/cgi-bin/models/get_mor.pl?key=6967725. Whether CJ0248 is still implicated in hydrolysis or signal transduction and how it functions in the virulence of Campylobacter jejuni is unclear and awaits further functional studies. The structure of CJ0248 from Campylobacter jejuni, determined by X-ray crystallography, represents a new fold. The information reported here, in combination with further biochemical and biophysical studies, should yield valuable insight into the functional role of this protein. This work was supported by the NIH Protein Structure Initiative from the National Institute of General Medical Sciences (www.nigms.nih.gov). Portions of this research were carried out at the Stanford Synchrotron Radiation Laboratory (SSRL) and the Advanced Light Source (ALS). The SSRL is a national user facility operated by Stanford University on behalf of the U.S. Department of Energy, Office of Basic Energy Sciences. The SSRL Structural Molecular Biology Program is supported by the Department of Energy, Office of Biological and Environmental Research, and by the National Institutes of Health (National Center for Research Resources, Biomedical Technology Program, and the National Institute of General Medical Sciences). The ALS is supported by the Director, Office of Science, Office of Basic Energy Sciences, Materials Sciences Division, of the U.S. Department of Energy under Contract No. DE-AC03-76SF00098 at Lawrence Berkeley National Laboratory.

Source: Proteins Structure Function and Bioinformatics

DOI: 10.1002/prot.20611