Crystal structure of an HEPN domain protein (TM0613) from <i>Thermotoga maritima</i> at 1.75 Å resolution

The TM0613 gene of Thermotoga maritima (Swissprot Q9WZ82) encodes a conserved hypothetical protein with a molecular weight of 13,964 Da and a calculated isoelectric point of 5.17. Currently, there is no functional annotation for this protein. On the genomic level, TM0613 is followed by a nucleotidyltransferase, TM0614, which is found on the same DNA strand, but in a different reading frame that partly overlaps the TM0613 reading frame. Such pairing of genes is observed in other species and believed to result in functionally-interacting proteins.1, 2 Here, we report the crystal structure of TM0613 determined using the semiautomated high-throughput pipeline of the Joint Center for Structural Genomics.3 The structure of TM0613 was determined to 1.75 Å resolution using the multiple-wavelength anomalous dispersion (MAD) method. Data collection, model, and refinement statistics are summarized in Table I. The final model includes one protein molecule (residues 1–79 and 86–123) and 129 water molecules in the asymmetric unit. No electron density was observed for residues 80 to 85. The Matthews' coefficient (Vm) for TM0613 is 1.9 Å3/Da and the estimated solvent content is 35.2%. The Ramachandran plot produced by PROCHECK 3.44 shows that 97.2 % of the residues are in the most favored regions, 2.8 % in additional allowed regions, and no residues in the generously or disallowed regions. The TM0613 monomer consists of a polypeptide chain of 123 amino acids composed of 5 α-helices arranged in an up-and-down helical bundle5 (Fig. 1). The total helical content is 72.7%. A disulfide bond is observed between Cys24 and Cys79 (Fig. 1). Density is present for an alternate conformation of Cys24, which indicates partial cleavage of the disulfide bond in the crystal. This flexibility may explain the missing density for the adjacent residues 80–85. The crystallographic packing in the TM0613 structure suggests that a dimer is the biologically-relevant oligomeric form with a buried surface area of 808 Å2 per monomer (Fig. 1B). The dimer interface is formed through interaction of helices η1 and η2, involving His10, His13, His17, Phe19, Trp22, Phe25, Gln29, and Lys33 (Fig. 1C). A: Ribbon diagram of Thermotoga maritima TM0613 color coded from N-terminus (blue) to C-terminus (red). Helices are numbered H1 to H5. The disulfide bond between Cys24 and Cys79 is rendered in ball and stick. The disordered region between residues 79 and 86 is depicted as a dashed line. B: The TM0613 dimer shown in ribbon representation. C: Diagram showing the secondary structure elements in TM0613 superimposed on its primary sequence. The residues corresponding to the dimer interface are marked with a black line, as well as the Cys-Cys disulfide. A structural similarity search, performed with the coordinates of TM0613 using the DALI server,6 revealed strong structural similarity to the C-terminal domain of kanamycin nucleotidyltransferase (KNTase; PDB: 1KNY). The RMSD between TM0613 and 1KNY is 2.9 Å over 98 aligned residues with 8% sequence identity. The KNTase contains two domains. The C-terminal domain is structurally similar to TM0613, whereas the N-terminal domain is homologous to TM0614. Interestingly, the KNTase contains a nucleotide binding site, involving residues from both domains. KNTases are an enzyme family responsible for conferring bacterial resistance to aminoglycoside antibiotics.7 Based on the similarity to KNTase and the localization of the TM0613 gene with respect to TM0614, we propose that the function of this conserved hypothetical protein is related to nucleotide binding. Another structural homolog is Hi0074 (PDB: 1JOG) from Haemophilus influenzae.2 The RMSD between TM0613 and Hi0074 is 3.0 Å over 104 aligned residues with 9.6% sequence identity. PSI-BLAST searches in public sequence databases revealed that TM0613 is a founding member of an emerging family of HEPN domain (Higher Eukaryotes and Prokaryotes NTP-binding domain) proteins,1 including more than 90 sequence homologs in Archaea, Bacteria, and higher eukaryotes. The human protein sacsin (Swissprot: Q9NZJ4) contains the HEPN domain at the C-terminus and is involved in the autosomal recessive early-onset neurodegenerative disease Spastic Ataxia of Charlevoix-Saguenay (SACS, OMIM 270550).8 The HEPN is believed to be truncated in mutations that cause the SACS disease. Models for a number of TM0613 homologs can be accessed at http://www1.jcsg.org/cgi-bin/models/get_mor.pl?key=TM0613. The TM0613 structure reported here represents the first example of a HEPN domain. We expect that the information that can be derived from this structure will yield valuable insights into the determinants of function and thermal stability of this protein family. TM0613 (TIGR: TM0613; SwissProt: Q9WZ82) was amplified by PCR from Thermotoga maritima strain MSB8 genomic DNA using PfuTurbo (Stratagene) and primer pairs encoding the predicted 5′- and 3′-ends of TM0613. The PCR product was cloned into plasmid pMH2T7, which encodes an expression and purification tag corresponding to residues MGSDKIHHHHHH at the amino terminus of the full-length protein. The cloning junctions were confirmed by sequencing. Protein expression was performed in 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 7.9, 50 mM NaCl, 0.25 mM Tri (2-carboxyethyl) phosphine hydrochloride (TCEP)) and cell debris pelleted by centrifugation at 3400 × g for 60 min. The soluble fraction was applied to a metal chelate affinity resin (Amersham Biosciences) previously charged with nickel and equilibrated with Equilibration Buffer (50 mM potassium phosphate pH 7.8, 0.25 mM TCEP, 10% v/v glycerol, 300 mM NaCl) containing 20 mM imidazole. The resin was washed with Equilibration Buffer containing 40 mM imidazole, and the protein eluted with Elution Buffer (20 mM Tris pH 7.9, 10% glycerol, 0.25 mM TCEP, 300 mM imidazole). Buffer exchange was performed to remove imidazole from the eluate, and the protein in Buffer Q (20 mM Tris pH 7.9, 5% v/v glycerol, 0.25 mM TCEP) containing 50 mM NaCl was applied to a Resource Q column (Amersham Biosciences) previously equilibrated with the same buffer. Protein was eluted using a linear gradient of 50 to 500 mM NaCl in Buffer Q, and appropriate fractions were pooled. Protein was buffer exchanged into size exclusion chromatography (SEC) Buffer (20 mM Tris pH 7.9, 150 mM NaCl, 0.25 mM TCEP) and concentrated for crystallization assays by centrifugal ultrafiltration (Millipore). The protein was crystallized using the nanodroplet vapor diffusion method9 with standard JCSG crystallization protocols.3 The crystallization solution contained 10% (w/v) PEG-8000 with 200 mM calcium acetate in 100 mM imidazole at pH 8.0. The crystals were indexed in the tetragonal space group P41212 (Table I). Multi-wavelength anomalous diffraction data were collected at ALS (Berkeley, USA) on beamline 5.0.3 (Table I). All data sets were collected at 100 K using a Quantum 4 CCD detector. Data were integrated and reduced using Mosflm10 and then scaled with the program SCALA from the CCP4 suite.11 Data statistics are summarized in Table I. The structure was determined using SOLVE12 and RESOLVE.13 Structure refinement was performed using CNS and REFMAC5.14, 11 The asymmetric unit contains one molecule. Refinement statistics are summarized in Table I. The final model for the protein contains the native structure (residues 1–79 and 86–123) plus residues His -2, -1, and 0 from the purification tag. Residues 80 to 85, as well as side chains for residues K5, K36, and R122, were disordered and, therefore, excluded from the model. Analysis of the stereochemical quality of the models was accomplished using the JCSG Validation Central suite, which integrates seven validation tools: Procheck 3.5.4, SFcheck 4.0, Prove 2.5.1, ERRAT, WASP, DDQ 2.0, and Whatcheck. The Validation Central suite is accessible at http://www.jcsg.org. Atomic coordinates of the final model and experimental structure factors of TM0613 have been deposited with the PDB and are accessible under the code 1o3u. Portions of this research were carried out at the Stanford Synchrotron Radiation Laboratory, 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).

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