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In the plant pathogen Xanthomonas campestris pv. campestris, the product of the tcmJ gene, XcTcmJ, encodes a protein belonging to the RmlC family of cupins. XcTcmJ was crystallized in a monoclinic space group (C2) in the presence of zinc acetate and the structure was determined to 1.6 Å resolution. Previously, the apo structure has been reported in the absence of any bound metal ion [Chin et al. (2006 ), Proteins, 65, 1046–1050]. The most significant difference between the apo structure and the structure of XcTcmJ described here is a reorganization of the binding site for zinc acetate, which was most likely acquired from the crystallization solution. This site is located in the conserved metal ion-binding domain at the putative active site of XcTcmJ. In addition, an acetate was also bound within coordination distance of the zinc. In order to accommodate this binding, rearrangement of a conserved histidine ligand is required as well as several nearby residues within and around the putative active site. These observations indicate that binding of zinc serves a functional role in this cupin protein.
The tcmJ gene (Shen & Hutchinson, 1993 ) of Xanthomonas campestris pv. campestris strain ATCC 33913 encodes a protein from the cupin superfamily, XcTcmJ, with a molecular weight of 12.2 kDa (residues 1–113) and a calculated isoelectric point of 4.9. XcTcmJ was selected for structure determination in order to extend the structural coverage of proteins of unknown function from pathogenic bacteria, in this case X. campestris pv. campestris (Leyns et al., 1984 ). This Gram-negative bacterium causes black rot, which is one of the major worldwide diseases of cruciferous crops (Crossman & Dow, 2004 ; Williams, 1980 ). In X. campestris pv. campestris, a variety of polyketide metabolites are synthesized which are precursors of antibiotics and antitumor therapeutics (Donadio et al., 2007 ; Tudor-Nelson et al., 2003 ). XcTcmJ is among several gene products from X. campestris that have been annotated as being involved in tetracenomycin polyketide biosynthesis (Overbeek et al., 2005 ). A sequence-database search found four proteins that are orthologous to XcTcmJ (Fig. 1 c), as well as over 1000 more distantly related proteins from several different protein families.
XcTcmJ belongs to the cupin superfamily, the members of which have a conserved β-barrel fold and have been classified into at least 18 functional classes involved in a range of biochemical processes, including plant growth and development (Dunwell et al., 2001 , 2004 ). In enzymatic cupins, the nature of the bound metal cofactor is believed to influence the type of reaction catalyzed. Although some characteristics of the metal ion-binding motif are conserved across all members of the cupin superfamily, several crystal structures of enzymatic cupins have been reported without any bound metal cofactor.
Comparison of apo and metal-bound cupin structures should, therefore, enhance our understanding of how metal-ion binding influences functional specificity in this superfamily of proteins. Since no metal ion was found in the previously solved crystal structure of an identical protein from a different strain (XC5357 from X. campestris pv. campestris strain 17; PDB code 2gu9; Chin et al., 2006 ), it was speculated that XC5357 may not require a metal ion for activity. Here, we report the crystal structure of XcTcmJ from X. campestris pv. campestris with zinc acetate bound at the putative active site at 1.6 Å resolution which offers additional insights into the role of metal ions in the structure and function of cupins. The structure was determined using the semi-automated high-throughput pipeline of the Joint Center for Structural Genomics (JCSG; Lesley et al., 2002 ) as part of the National Institute of General Medical Sciences Protein Structure Initiative (PSI; http://www.nigms.nih.gov/Initiatives/PSI/).
The gene encoding XcTcmJ (GenBank NP_636471.1; gi:21230554) was amplified by polymerase chain reaction (PCR) from genomic DNA using PfuTurbo DNA polymerase (Stratagene) and primers corresponding to the predicted 5′ (aacctgtacttccagggcATGCAGTACGCAACGTTGGA) and 3′ (gagttaattaagtcgcgttaGCCTTCGCCTGCCGGCAA) ends. The PCR product was cloned into plasmid pSpeedET, which encodes an expression and purification tag followed by a tobacco etch virus (TEV) protease cleavage site (MGSDKIHHHHHHENLYFQ/G) at the amino-terminus of the full-length protein. The cloning junctions were confirmed by DNA sequencing. Protein expression was performed in selenomethionine-containing medium with suppression of normal methionine synthesis using Escherichia coli strain GeneHogs (Invitrogen). At the end of fermentation, lysozyme was added to the culture to a final concentration of 250 µg ml−1 and the cells were harvested. After one freeze–thaw cycle, the cells were sonicated in lysis buffer [50 mM HEPES pH 8.0, 50 mM NaCl, 10 mM imidazole, 1 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP)]. The lysate was clarified by centrifugation at 32 500g for 30 min and loaded onto nickel-chelating resin (GE Healthcare) pre-equilibrated with lysis buffer. The resin was washed with wash buffer [50 mM HEPES pH 8.0, 300 mM NaCl, 40 mM imidazole, 10%(v/v) glycerol, 1 mM TCEP] and the protein was eluted with elution buffer [20 mM HEPES pH 8.0, 300 mM imidazole, 10%(v/v) glycerol, 1 mM TCEP]. The eluate was buffer-exchanged with HEPES crystallization buffer (20 mM HEPES pH 8.0, 200 mM NaCl, 40 mM imidazole, 1 mM TCEP) and treated with 1 mg of TEV protease per 15 mg of eluted protein. The digested protein was passed over nickel-chelating resin (GE Healthcare) pre-equilibrated with HEPES crystallization buffer and the resin was washed with the same buffer. The flowthrough and wash fractions were combined and concentrated to 18 mg ml−1 by centrifugal ultrafiltration (Millipore) for crystallization assays. XcTcmJ was crystallized using the nanodroplet vapor-diffusion method (Santarsiero et al., 2002 ) with standard JCSG crystallization protocols (Lesley et al., 2002 ). Sitting drops composed of 200 nl protein mixed with 200 nl crystallization solution were equilibrated against a 50 µl reservoir at 277 K for 34 d prior to harvesting. Initial screening for diffraction was carried out using the Stanford Automated Mounting system (SAM; http://smb.slac.stanford.edu/facilities/hardware/SAM/UserInfo; Cohen et al., 2002 ) at the Stanford Synchrotron Radiation Lightsource (SSRL). The crystallization reagent contained 35%(v/v) 2-propanol, 0.2 M zinc acetate and 0.1 M imidazole pH 8.0. No additional cryoprotectant was added to the crystal. Diffraction data from a cube-shaped crystal of approximate dimensions 100 × 100 × 100 µm mounted in a nylon loop were indexed in monoclinic space group C2 (Table 1 ). The molecular weight and oligomeric state of XcTcmJ were determined using a 1 × 30 cm Superdex 200 column (GE Healthcare) in combination with static light scattering (Wyatt Technology). The mobile phase consisted of 20 mM Tris pH 8.0, 150 mM NaCl and 0.02%(w/v) sodium azide.
Multi-wavelength anomalous diffraction (MAD) data were collected at SSRL on beamline BL11-1 at wavelengths corresponding to the high-energy remote (λ1), inflection (λ2) and peak (λ3) of a selenium MAD experiment. The data sets were collected at 100 K using an ADSC Quantum 315 CCD detector. The MAD data were integrated and scaled using the XDS package (Kabsch, 1993 ). Data statistics are summarized in Table 1 . The selenium substructure was solved with SHELXD (Sheldrick, 2008 ) and the MAD phases were refined with autoSHARP (Vonrhein et al., 2007 ), which resulted in a mean figure of merit of 0.42 with two selenium sites. Automated model building was performed by iterative cycles of ARP/wARP (Cohen et al., 2004 ). Model completion and refinement were performed using Coot (Emsley & Cowtan, 2004 ) and REFMAC (Winn et al., 2003 ). Refinement statistics are summarized in Table 1 .
The quality of the crystal structure was analyzed using the JCSG Quality Control server, which verifies the stereochemical quality of the model using AutoDepInputTool (Yang et al., 2004 ), MolProbity (Lovell et al., 2003 ) and WHATIF 5.0 (Vriend, 1990 ), the agreement between the atomic model and the data using SFCHECK 4.0 (Laskowski et al., 2005 ) and RESOLVE (Terwilliger, 2002 ), the protein sequence using ClustalW (Chenna et al., 2003 ), the atom occupancies using MOLEMAN2 (Kleywegt et al., 2001a ,b ) and the consistency of NCS pairs. Protein quaternary-structure analysis used the EBI PISA server (Krissinel & Henrick, 2007 ). Fig. 1 (b) was adapted from an analysis using PDBsum (Laskowski et al., 2005 ). Figs. 1 (a) and 2 (a) were prepared with PyMOL (DeLano Scientific) and Fig. 1 (c) was prepared with ESPript (Gouet et al., 2003 ). Atomic coordinates and experimental structure factors for XcTcmJ from X. campestris pv. campestris strain ATCC 33913 at 1.6 Å resolution have been deposited in the PDB under accession code 3h50.
The crystal structure of XcTcmJ (Fig. 1 a) was determined to 1.6 Å resolution using the MAD method. Data collection, model and refinement statistics are summarized in Table 1 . The final model is comprised of a protein monomer (residues 1–113 and Gly0 from the TEV protease cleavage site), four zinc ions, two acetate molecules and 151 water molecules in the asymmetric unit. As the electron density was not well resolved for some of the side-chain atoms of Lys13, Gln20 and Arg42, these atoms were not included in the structural model. The Matthews coefficient (V M; Matthews, 1968 ) for XcTcmJ is 2.19 Å3 Da−1 and the estimated solvent content is 43.8%. The Ramachandran plot produced by MolProbity shows that 98.2% and 100% of the residues are in favored and allowed regions, respectively.
XcTcmJ is composed of ten β-strands (β1–β10) and one 310-helix (Fig. 1 a). The total β-sheet content is 45.1% and the 310-helical content is 2.6%. The XcTcmJ monomer (Fig. 1 a) contains two antiparallel β-sheets that form a jelly-roll β-sandwich and a topology characteristic of the RmlC-like cupin superfamily (SCOP sunid 51182; Andreeva et al., 2004 ). The larger β-sheet has a six-stranded 23(10)581′ topology, while the smaller β-sheet has a four-stranded 4967 topology (Fig. 1 a).
The crystallographic packing of XcTcmJ indicates that a homodimer is the biologically relevant form. This finding is consistent with previous reports (Chin et al., 2006 ; Chu et al., 2005 ) and with the results of our analytical size-exclusion chromatography coupled with static light scattering. In the homodimer, the N-terminal β-strand β1 of each monomer interacts with β8 in the twofold symmetry-related monomer in a domain-swapped arrangement. The dimer resembles two juxtaposed barrels. One end of each barrel, proximal to the C-terminus and zinc-binding site, is solvent accessible and forms a putative active site. The two symmetry-related, six-stranded β-sheets in the dimer show a pronounced twist, which enhances structural complementarity to the tightly packed, monomer–monomer interface.
A DALI search (Holm et al., 2008 ) for entries in the PDB similar to XcTcmJ revealed a multitude of related cupin structures. Alignment of the amino-acid sequence of XcTcmJ with those of related structures is shown in Fig. 1 (c). In general, XcTcmJ shares only 10–20% sequence identity with related cupins, even though the structures are quite similar. Many structural homologues contain a bound metal ion, but others do not. An example from the multitude of similar structures is the metal-deficient BH2720 from Bacillus halodurans (gi:10175341; PDB code 2oa2), which was reported by the JCSG and which has 28% sequence identity and an r.m.s.d. of 1.46 Å with XcTcmJ for 99 superimposed Cα atoms.
X-ray fluorescence emission spectroscopy on the same XcTcmJ crystal as used for structure determination indicated the presence of selenium and zinc and the absence of other transition metals. To corroborate that zinc is bound at specific sites in the structure and is not just present in the bulk solvent, an anomalous difference map was calculated using the Se remote data (λ1 MADSe in Table 1 ). The map revealed that, in addition to two electron-dense peaks ~16σ above the r.m.s. background level at positions corresponding to the Se atoms, other peaks at ~13σ were found that were not associated with the selenium sites. These peak-height ratios were consistent with the theoretical ratio of Δf′′ for selenium versus zinc at λ1. One of the bound zinc ions is specifically coordinated to the metal-binding site (Figs. 1 a and 2 ), while three other zinc ions are found in crystal-packing interfaces (not shown). The active-site zinc is within coordination distance of the side-chain NE2 atoms of two histidines (His41 and His80) and the carboxamide O atom of Gln46. A bound acetate was also identified in an ~5 Å deep hydrophobic pocket and was most likely acquired from the crystallization solution. The positions of the acetate carboxylate emulates coordination to the zinc by Glu or Asp side chains in other structures. Here, the Glu34 carboxyl is 5 Å from the bound acetate and is too distant for zinc coordination even if it adopted another rotamer (Fig. 2 b).
A bound water molecule, acetate and three side-chain ligands indicate octahedral coordination to the bound zinc. In zinc metalloproteins the metal ion is thought to play either a structural or a catalytic role, with tetrahedral coordination being the most prevalent (McCall et al., 2000 ). Structural zinc sites usually contain four protein side-chain ligands (Cys, Glu or Asp) in a tetrahedral coordination, whereas catalytic zinc sites tend to utilize more than four ligands, with one being a water (Auld, 2009 ). Thus, the octahedral coordination geometry and coordinating water molecule both suggest a likely functional role for the bound zinc. A list of interactions surrounding the zinc at the putative active site is shown in Table 2 .
Most members of the cupin superfamily typically share two conserved sequence motifs (Dunwell et al., 2001 , 2004 ; see Fig. 1 c) that contribute His, Glu and Asp side chains to a conserved metal ion-binding site. An alignment between XcTcmJ and related sequences is shown in Fig. 1 (c), with the conserved N-terminal sequence motif highlighted in green and the C-terminal sequence motif in blue. This alignment indicates that, in XcTcmJ, either Asp38 or Asp45 is likely to be a ligand for a bound metal ion. However, the crystal structure shows that the ligand of the metal ion is the carboxamide side chain of Gln46, even though Glu and Asp side chains are more prevalent ligands than the carboxamide side chains of Gln and Asn residues (Hsin et al., 2008 ). This type of coordination is also observed in the crystal structure of a related cupin, phosphomannose isomerase from Candida albicans (Cleasby et al., 1996 ), where an amide side chain is also within coordination distance of the bound metal.
Apo XcTcmJ has previously been reported at 1.4 Å resolution for XC5357 from a different strain of X. campestris (PDB code 2gu9; Chin et al., 2006 ). Although XC5357 and XcTcmJ share identical amino-acid sequences, their structures show significant differences. XC5357 was crystallized in a triclinic form without zinc, whereas XcTcmJ crystallized in a monoclinic space group in the presence of zinc acetate. Examination of the structures with and without zinc reveals significant rearrangements around the metal-binding site (Figs. 3 a and 3 b). Strand β4 (Gly35–Asp38) in the apo structure is repositioned as a surface loop in the XcTcmJ structure. Additional rearrangements (Asn39–Gly43) in XcTcmJ contribute to the formation of a longer loop segment connecting β4 (Asp32–Gly36) and β5 (Asp45–Asp52) at the open end of the cupin barrel. The conserved His41 is located in one of the loops and the backbone rearrangements in XcTcmJ result in His41 relocating by ~8 Å to coordinate with the Zn atom near the center of the cupin barrel. In contrast, the other zinc ligands, Gln46 and His80, do not move significantly.
Comparison of the solvent-accessible surfaces (see supplementary material1) indicates that a deep surface cavity is eliminated upon the conformational rearrangements arising from the binding of zinc acetate. In the apo structure, this cavity is located between antiparallel β-strands at the C-terminus of each monomer and a loop in the vicinity of His41. In XcTcmJ, this histidine and loop are repositioned upon metal ligation, so that the interactions with the C-terminal region are lost. One possible explanation for the loss of these interactions is that the crystallized XC5357 contained an eight-residue sequence tag at the C-terminus (Chu et al., 2005 ) that was absent from the crystallized XcTcmJ. Consequently, this cavity is replaced by a shallow depression in XcTcmJ and the two-stranded β-sheet becomes a loop and a small 310 helix. Notwithstanding, both the XcTcmJ and apo structures retain the cavity corresponding to the zinc acetate-binding site. Furthermore, a new channel is formed on the surface of XcTcmJ that is surrounded by nonpolar residues including Ala11, Phe12, Val27 and Ile28 that may reflect the natural substrate-binding site.
Extensive investigations have demonstrated the importance of metal ions in the structure and function of diverse cupins (Dunwell et al., 2001 , 2004 ). However, it is not understood why some cupins require metal ions for biological activity whereas others do not. Conformational changes induced by metal binding could exert control of reaction specificity or even inhibition of certain biological processes (Babor et al., 2005 ; Barondeau & Getzoff, 2004 ). From the structural studies alone, it is difficult to define the exact functional implications of metal ion binding to XcTcmJ. However, metal ion binding can have a profound impact on the structure, as illustrated here. Zinc is specifically bound and reorganizes the structure and acetate (likely from the crystallization solution) then serves as a counter ion in the formation of the zinc–protein complex.
Additional information about the protein described in this study is available from TOPSAN (Krishna et al., 2010 ) http://www.topsan.org/explore?PDBid=3h50.
PDB reference: tetracenomycin polyketide synthesis protein (NP_636471.1), 3h50, r3h50sf
This work was supported by the National Institutes of Health, Protein Structure Initiative (http://www.nigms.nih.gov), grant Nos. P50 GM62411 and U54 GM074898. Portions of this research were carried out at the Stanford Synchrotron Radiation Lightsource (SSRL). The SSRL is a national user facility operated by Stanford University on behalf of the US 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 content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of General Medical Sciences or the National Institutes of Health.
1Supplementary material has been deposited in the IUCr electronic archive (Reference: WD5112).