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1 Protein Structure Factory, Berlin 12489, Germany
2 Max-Delbrück-Centrum für Molekulare Medizin, Berlin 13092, Germany
3 Institut für Chemie und Biochemie, Berlin 14195, Germany
4 Protein Structure Factory, Berlin 14059, Germany
5 Max-Planck-Institut für Molekulare Genetik, Berlin 14195, Germany
6 Institut für Medizinische Physik und Biophysik, Charité Universitätsmedizin Berlin, Berlin 10098, Germany
(RECEIVED December 13, 2005; FINAL REVISION December 13, 2005; ACCEPTED December 16, 2005)
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four-layer topology. A metal ion residing between the central -sheets is partially coordinated by three histidine residues. X-ray absorption near-edge structure (XANES) analysis identifies the PTD012-bound ion as Zn2+. Tetrahedral coordination of the ion is completed by the carboxylate oxygen atom of an acetate molecule taken up from the crystallization buffer. The binding of Zn2+ to PTD012 is reminiscent of zinc-containing enzymes such as carboxypeptidase, carbonic anhydrase, and -lactamase. Biochemical assays failed to demonstrate any of these enzyme activities in PTD012. However, PTD012 exhibits ester hydrolase activity on the substrate p-nitrophenyl acetate. Keywords: PTD012 family; alternative splicing; splice variant; structural genomics; Zn-binding site; ester hydrolase
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Introduction |
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Homologous sequences to PTD012 have been found in Mus musculus (SwissProt Q91V76, 315 residues, 87% sequence identity), Brachydanio rerio (Q6PHI5, 318, 62%), Drosophila melanogaster (Q9W0J9,322,47%; Q8IRI0,361, 44%; Q8SXQ8, 322, 44%), Anopheles gambiae str. PEST (Q7QI91, 360, 41%), and Caenorhabditis elegans (Q18131, 192, 36%). Figure 1A shows an alignment of these proteins with PTD012 (splice variant Q9H0W9).
In humans, ~30%–60% of the genes undergo alternative splicing (Lander et al. 2001; Venter et al. 2001; Garcia-Blanco et al. 2004) and, in some cases, splice variants are associated with human diseases. The ENSEMBL database (Hubbard et al. 2002) lists a PTD012 splice variant corresponding to a 315-residue protein (SwissProt Q9H0W9) and a second variant lacking 50 amino acids in the central region (Q96EI3). The sub-cellular location of PTD012 was revealed as predominantly nuclear by an automatic phenotyping approach (Conrad et al. 2004); see LIFEdb database (Bannasch et al. 2004). Neither the biochemical function nor the biological role of PTD012 or any of its homologs are known.
Here we report biophysical data (see Supplemental Material) and the crystal structure of the long PTD012 splice form determined by SAD phasing. The protein adopts a zinc-containing hydrolase fold. Zn2+ is tetrahedrally coordinated to imidazole nitrogens of three histidine residues and to a carboxylate oxygen of an acetate anion taken up from the crystallization buffer in a way reminiscent of Zn2+-dependent hydrolases. PTD012 exhibits esterase activity toward synthetic substrates.
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Results and Discussion |
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positions for residues 3 to 315 of 0.27 Å. The solvent accessible surface buried between the two monomers is 633 Å2, corresponding to 5.2% of the total surface area (12,120 Å2 per molecule). Since the fraction of surface area buried in dimers tends to be significantly larger (Jones and Thornton 1995), we conclude that PTD012 exists as a monomer in solution.
Each PTD012 molecule is a single polypeptide chain, composed of 315 residues and roughly globular in shape with a diameter of ~45 Å. The overall structure is mainly composed of 20 -strands and 4 -helices which are numbered as shown in Figure 1B. The secondary structure assignment was made using PROMOTIF (Hutchinson and Thornton 1996). The PTD012 structure is organized into a four-layer topology. Structure-based searches using DALI (Holm and Sander 1993) and VAST (Gibrat et al. 1996) did not point to any homologous structures in the Protein Data Bank. Distant structural homology appears to exist with PDB entry 1XV2, a Staphylococcus aureus protein sharing sequence similarity with -acetolactate decarboxylase (A. Murzin, pers. comm.). Even though it is a four-layer sandwich, the topology of the PTD012 monomer does not fall under any existing classification of four-layer structures (Carfi et al. 1995; Guddat et al. 1999; Knöfel and Sträter 1999; Kumaran et al. 2003), since the order of -strands, the connectivity, and crossovers of the sheets are highly different. The shorter splice variant, which lacks the central 50 residues, is expressed strongly in Escherichia coli but is completely insoluble. The PTD012 structure suggests that a protein lacking these 50 residues cannot fold and is likely non-functional.
Nine antiparallel -strands in the order 2, 12, 19, 13, 16, 17, 18, 14, and 15 form a -screw architecture which is rolling around helix 3, and the rest of the 11 mixed -strands form a -layer, which is partially twisted adjacent to helix 2. The interface of the -screw and -layer divides the molecule into two halves. However, the connecting loops between them have filled up the empty gaps. In addition, the helices 1, 4, and two 310 helices are flanking the surface of the molecule. A further database search was carried out with VAST (Gibrat et al. 1996) to identify structural neighbors of PTD012 substructures. For the -layer region (aa 51–165), the search did not yield any structural neighbors. However, a search using the -screw region of PTD012 (aa 171–296) revealed structural similarity to the cyclic adenosine 3',5'-monophosphate (cAMP) binding domain of the regulatory subunit of protein kinase A (1GRS, 53 matched residues out of 125, RMSD = 2.7 Å, Z-score 7.8, 13% sequence identity) (Su et al. 1995; Wu et al. 2004). The superposition of the matched regions is shown in Figure 1C. In addition to the conservation of much of the secondary structure, we note that the cAMP-binding site of the PKA regulatory subunit superimposes onto the Zn2+-binding site of PTD012 (see below).
Cation-binding site
Difference Fourier maps revealed strong electron density at the -screw/-layer interface within coordination distance (~2 Å) to atoms N
of H278, N
of H266 and N of H268, suggesting that a metal atom is bound to the protein at an HxHxxxxxxxxxH motif (Fig. 2A). The metal ion bound to PTD012 was determined to be zinc. In a fluorescence spectrum scan (h
= 9670 eV) the X-ray emission (fluorescence) line K of Zn and the elastic scattered line were clearly resolved (Fig. 2B). Furthermore, the X-ray absorption near-edge structure (XANES) could be recorded by taking the counts solely in the region where the Zn K line is located. Moreover, the excitation photon energy was scanned through the XANES energy range of the Zn K-edge (Fig. 2C). In the structure, the tetrahedral Zn coordination is completed with density likely to represent an acetate molecule. Acetate may have been recruited from the crystallization buffer in which it was present at a 200-mM concentration. Occupation of the fourth coordination site by a molecule is very suggestive of an enzymatic role for the metal ion, as Zn2+ is similarly coordinated in many zinc-containing enzymes (Lipscomb and Sträter 1996; Hayashi 2004).
Comparison with Zn-containing enzymes
The structure of PTD012 exhibits striking similarities with the structure of -carbonic anhydrase (-CA) (Tripp et al. 2001), even though no sequence homology is found between them. First, the dominating secondary structure is a 10-stranded, twisted -sheet. Second, an HxH motif is utilized in zinc coordination. In addition to this conservation, the carboxylate group of residue E110 resides 3.8 Å away from the Zn2+ ion (5 Å away in -CA). Furthermore, an acetate molecule (structurally analogous to bicarbonate) is bound in the crystal structure of PTD012, resembling one of the reaction products within the pathway of reversible hydration of carbon dioxide to carbonic acid (or to bicarbonate), which is catalyzed by -CA (Liljas and Laurberg 2000; Tripp et al. 2001). In view of this, an assay for carbonic anhydrase activity was performed (Supplemental Material). In this assay, PTD012 did not show carbonic anhydrase activity, suggesting that PTD012 does not belong to the -CA family.
The arrangement of residues R263 and E110 near the cation-binding site of PTD012 is very similar to the arrangement of R482 and E626 near the active site of human carboxypeptidase A2 (CPA2) (Reverter et al. 2000). In CPA2, the residue E626 (E110 of PTD012) acts as a general acidic catalyst to form a metal-bound hydroxide species (zinc hydroxide). The zinc hydroxide then acts as the nucleophile to attack the peptide carbonyl function that is polarized with the assistance of R482 (R263 in PTD012). In addition to the conservation of these two residues in PTD012, the carboxylate group of the PTD012 C terminus resides very close to the cation-binding site. One of the C-terminal carboxylate oxygens of E315 interacts with the imidazole N of the Zn2+-coordinating residue H278 within a distance of 2.8 Å, and the other C-terminal oxygen of E315 forms a hydrogen bond with the guanidinum group of R263 (over 3.3 Å). Interestingly, the amino acid sequence at the C terminus, a GRD motif, is well conserved in all PTD012 homologs (Fig. 1A). Moreover, the mode of binding of the acetate carboxylate group to the zinc ion is reminiscent of the binding of the leech carboxypeptidase inhibitor (LCI) C terminus to the human CPA2 active site (Reverter et al. 2000). In view of the conservation of residues R263 and E110 near the cation-binding site, the binding of the fourth ligand (acetate), and the interaction between the C termini and the active site, a possible carboxypeptidase activity was tested by incubating a number of peptides with the protein, followed by mass spectrometric analysis of the reaction mixtures (see Supplemental Material). No proteolytic activity was observed on any of the standard peptides, ruling out the possibility that PTD012 might belong to the carboxypeptidase family of enzymes.
PTD012 and -lactamase show similar four-layered sandwich structures and Zn2+-binding sites at the interface of two sheets, although the overall structural topology between the two proteins is rather poor. -lactamase utilizes Zn2+ to hydrolyze a series of -lactam antibiotics, such as penicillin and cephalosporin derivatives (Massova and Mobashery 1999). A hydrolytic activity of PTD012 on ampicillin as substrate was not detectable. However, when the ester hydrolase activity of PTD012 was assayed with the substrate p-nitrophenyl acetate (pNPAc), this compound was hydrolyzed to nitrophenolate and acetate. The crystal structure showing acetate bound to the Zn2+-binding site of PTD012 may thus represent an enzyme-product complex. The enzymatic turnover number for PTD012 was 0.042 sec–1. In comparison, for bovine carbonic anhydrase II, which also catalyzes the ester hydrolysis of pNPAc, a turnover number of 1 sec–1 was determined. Therefore, PTD012 is an ester hydrolase and might belong to the superfamily of metallo--lactamase fold proteins.
Conclusion
The long splice variant of human PTD012 is a monomeric protein with a novel overall fold. The shorter transcript does not seem to encode a folded protein. PTD012 contains a Zn2+ ion coordinated to three histidines and an acetate anion at a site that likely supports the enzymatic activity of the protein as an ester hydrolase. The anion binding to the putative active site may prove useful in studies of the properties of the metal center, particularly the search for the natural substrate and various specific inhibitors to derive the enzymatic mechanism and to understand the underlying biological function.
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Materials and Methods |
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Fermentation and cultivation ofE. coli clones
Clone 110,535 was grown at 37°C to an OD600 of 8.5 in SB medium (12 g/L Bacto-tryptone, 24 g/L yeast extract, 0.4% (v/v) glycerol, 17 mM KH2PO4 ,72 mM K2HPO4), supplemented with 100 µg/mL ampicillin and 34 µg/mL chloramphenicol in shaker flasks. Gene expression was induced by addition of 1 mM isopropyl--D-thiogalactopyranoside (IPTG). After 3.5 h the cells were harvested by centrifugation (6000g, 15 min, 4°C) and stored at –80°C. Clone 111491 was expressed using an inhouse protocol mainly based on the stepwise cultivation method of Budisa et al. (1995) (see Supplemental Material).
Protein purification
Cell pastes of clones 110535 (unlabeled) and 111491 (SeMet-labeled) were resuspended in extraction buffer (20 mM Tris-HCl at pH 8.0, 300 mM NaCl, 0.5 mM EDTA, 1 mM PMSF) containing lysozyme and benzonase, and either 4 mM or 10 mM 2-mercaptoethanol, respectively. The cells were disrupted by ultrasonic treatment in a chilling water bath, using a Bandelin Sonoplus sonifier. They were centrifuged 20 or 45 min at 55,000g and filtered using 0.45 µm syringe filters. The pH of the filtrate was adjusted to pH 7.4 and then applied to a 10-mL TALON column, pre-equilibrated with buffer (20 mM Tris-HCl at pH 7.4, 50 mM NaCl, 10 mM imidazole). After thorough washing, proteins were eluted with 200 mM imidazole in 20 mM Tris-HCl, 50 mM NaCl (pH 7.4). After removal of the His6 tag by overnight incubation with TEV protease at 4°C, protein solutions were subjected to POROS 20 S cation-exchange chromatography on a column pre-equilibrated with 20 mM Tris-HCl buffer, 15 mM NaCl (pH 7.4). Upon application of a linear NaCl gradient the PTD012 proteins were eluted separately from the TEV protease. The proteins were further purified on a POROS HQ anion-exchange column under the same buffer conditions, concentrated using Amicon Ultra-15 PLTK centrifugal filter units (10 kDa cut-off), and applied to a Superdex 75 column equilibrated with buffer (15 mM Tris-HCl at pH 7.4, 50 mM NaCl, 0.1 mM EDTA, 0.02% NaN3, and 2 or 5 mM DTT). For crystallization, the proteins were concentrated to 32 mg/mL each.
Esterase assay
Esterase activities were measured according to Rozema and Gellman (1996) using p-nitrophenyl acetate (pNPAc) as substrate. Samples containing 20 mg of enzyme were pipetted into 20 mM Tris-HCl (pH 7.7) to give a final volume of 400 µL. After addition of 45 µL of 52 mM pNPAc in dry acetonitrile and mixing, the formation of the hydrolysis product, p-nitrophenolate (400nm = 19,300 M–1cm–1), was monitored at room temperature by measuring the linear increase in absorbance at 400 nm between 30 and 60 sec (background hydrolysis was subtracted).
Crystallization and Data Collection
SeMet-derivatized crystals were grown from a protein solution at 32 mg/mL containing 20% PEG 4000, 0.2 M ammonium acetate, 0.1 M bis-Tris (pH 5.3) at 20°C. The crystals belong to space group P21 with cell parameters a = 44.5 Å, b = 52.9 Å, c = 123.5 Å, and = 90.8°. Native crystals were grown from a protein solution at 31.8 mg/mL containing 25% PEG 4000, 0.2 M ammonium acetate, 12% glycerol, 0.1 M Na acetate (pH 4.6), at 20°C. Also in P21, their cell parameters are a = 44.6 Å, b = 52.9 Å, c = 124.8 Å, = 88.9°. For both protein forms, the solvent content was calculated to 41% (VM = 2.1 Å3/Da) (Matthews 1968) assuming two molecules with a total molecular mass of 2 x 35,117 Da in the asymmetric unit. Single-wavelength anomalous diffraction data at the Se edge were collected at 100 K to 2.0 Å resolution on the SeMet-derivatized crystals and additionally high-resolution 1.7 Å native data were collected on the MAR345 imaging plate detector at beam-line PSF-ID14.2, BESSY, Berlin. The data sets were processed and scaled using the program HKL2000 (Otwinowski and Minor 1997). The data collection statistics are as shown in Table 1.
Phasing, model building, and refinement
The 12 selenium sites corresponding to 2 PTD012 molecules in the asymmetric unit were identified, refined, and phases were calculated using SOLVE (Terwilliger and Berendzen 1999). Solvent flattening and automatic model building using RESOLVE (Terwilliger 2001) allowed to trace 564 out of 630 residues. For phase extension to 1.7 Å resolution, the partial model was placed into the native unit cell by molecular replacement using MOLREP (Vagin and Teplyakov 2000) of the CCP4 suite (Collaborative Computational Project Number 4 1994). The rest of the residues were built manually with O (Jones et al. 1991), and REMAC5 (Murshudov et al. 1997) was employed to refine the model. Solvent molecules were located using the ARP/wARP suite (Morris et al. 2003). TLS parameters (Winn et al. 2001) were determined and refined for two molecules in the asymmetric unit. The final model consists of 626 residues, two zinc ions, two acetate, and 310 water molecules. The final refinement statistics are shown in Table 1. The stereochemical parameters of the protein model were evaluated with PROCHECK (Laskowski et al. 1993). The atomic coordinates and structure factors are available from the Protein Data Bank under accession code 1XCR. Figures were drawn with MOLSCRIPT (Kraulis 1991) and PYMOL (DeLano 2003).
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Footnotes |
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Supplemental material see www.proteinscience.org
Reprint requests to: Udo Heinemann, Max Delbrück Center for Molecular Medicine Robert-Rössle-Str. 10 D-13092, Berlin, Germany; e-mail: heinemann{at}mdc-berlin.de; fax: + 49-30-9406-2548.
Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.052037006.
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contour level in ochre. (B) Fluorescence spectrum at a photon energy of 9670 eV, taken by an Amptek-detector XR-100CR. (C) X-ray absorption near-edge structure (XANES) at the Zn K