Crystal structure of a putative PII‐like signaling protein (TM0021) from Thermotoga maritima at 2.5 Å resolution (2004)

The TM0021 gene of Thermotoga maritima encodes a conserved hypothetical protein with a molecular weight of 11,804 Da (residues 1–102) and a calculated isoelectric point of 6.9. Currently, there is no functional annotation for this protein. Here, we report the crystal structure of TM0021 in complex with adenosine diphosphate (ADP) that was determined using the semiautomated high-throughput pipeline of the Joint Center for Structural Genomics (JCSG).1 Based on the structure of the TM0021/ADP complex and its similarity to the signal transduction protein PII from Escherichia coli [Protein Data Bank (PDB) code: 2pii],2 we propose that TM0021 functions as a signal transducer in the regulation of the nitrogen status in T. maritima.3 The structure of TM0021 [Fig. 1(A)] was determined to 2.5 Å resolution using the single-wavelength anomalous dispersion (SAD) method. Data collection, model, and refinement statistics are summarized in Table I. The final model includes one protein molecule (residues 1–46 and 60–100 plus 2 N-terminal His-tag residues), 1 endogenous ADP molecule, 2 sulphates, and 52 water molecules. No electron density was observed for residues 47–59 and 101–102. The Matthews coefficient (Vm) for TM0021 is 3.79 Å3/Da, and the estimated solvent content is 67.3%. The Ramachandran plot produced by PROCHECK 3.44 shows that 95% of the residues are in the most favored regions, and 5% in additional allowed regions. Crystal structure of TM0021. (A) Ribbon diagram of Thermotoga maritima TM0021 color coded from N-terminus (blue) to C-terminus (red) showing the domain organization and the location of the ADP binding site. The ADP molecule is shown in ball and stick. α-helices (H1 and H2), β-sheet A (β1, β6, β5, β4) and β-sheet B (β2, β3) are indicated. The disordered T-loop is depicted with a dashed line. (B) Ribbon diagram of the TM0021 trimer with the bound ADP molecules shown in CPK mode. Figures produced with PYMOL (DeLano Scientific LLC). The final model of the TM0021 monomer consists of a single polypeptide chain of 88 amino acids containing 2 α-helices (H1 and H2) and 6 β-strands (β1–β6). The total α-helix and β-strand content is 25.8% and 38.2%, respectively. The TM0021 structure folds into an α+β sandwich with a 4-stranded antiparallel β-sheet (β1, β4, β5, β6 with 2X, -1, -2X topology) that packs against 2 antiparallel α-helices similar to the ferredoxin-like fold5 [Fig. 1(A)]. A short β-hairpin (β2, β3; residues 13–18) is inserted before the first α-helix (H1). The two surface-exposed loops correspond to the T-loop (residues 46–60) and the B-loop (residues 85–90) in PII proteins. The flexible T-loop, or recognition loop, is involved in protein–protein interactions with receptor molecules and is usually disordered in unliganded PII-like protein structures.2 TM0021 forms a tight trimer, with an inner channel, constructed by the orthogonal packing of its β-sheets around the crystallographic three-fold axis [Fig. 1(B)]. The B- and T-loops sit right at the edge of the β-barrel and protrude from the top of the molecule. The adenosine triphosphate (ATP) binding site is found next to the B-loop, at the junction of two adjacent β-sheets, that come from neighboring subunits of the trimer. The sequence motifs 43-GFGH-46 and 87-FDGL-90 (bold residues entirely conserved) constitute the ATP binding site [Fig. 2(A)]. The 43-GFGH-46 motif is located right before the T-loop, suggesting that ATP binding might have an effect on the conformation of the flexible T-loop. As no density was visible for a γ-phosphate of an ATP, the bound ligand was modeled as an ADP molecule. The interactions between ADP and its coordinating residues are depicted in Figure 2(B). The phosphate groups form hydrogen bonds to Gly45-N, His46-ND1, and Gly89-N. The sugar ring is stabilized by hydrophobic interactions with Tyr7 and Phe44, and a hydrogen bond to Val35-O and Thr36-OG1 from the neighboring subunit in the trimer. The adenine moiety is buried in a cleft between adjacent subunits and forms hydrogen bonds to Val69-O, Val69-N, and Thr36-OG1, and hydrophobic interactions with Gly34, which are located in the neighboring molecule. The ADP molecule must be derived from the bacterial expression system, because it was not present in the purification or crystallization buffers. (A) Diagram showing the secondary structure elements in TM0021 superimposed on its primary sequence. The residues involved in ADP binding are marked above with a black line. Residues corresponding to the T-loop (46–60) and the B-loop (85–90) are underlined. β-hairpins are depicted in red. Produced with PDB sum (http://www.biochem.ucl.ac.uk/bsm/pdbsum). (B) Schematic representation of the interactions between ADP and its coordinating residues. The ADP molecule is depicted in purple and the protein residues are in orange. The atoms are indicated as follows: carbon (black), oxygen (red), nitrogen (blue), and phosphor (purple). Hydrogen bonds are represented as dashed green lines. Residues implicated in hydrophobic interactions are represented as barbed circle sections. Figure produced with LIGPLOT.16 A structural similarity search, performed with the coordinates of TM0021 using the DALI server,6 indicates that the closest structural homologue is the signal transduction protein PII (product of glnB) from E. coli (PDB code: 2pii).2 The root-mean-square deviation (RMSD) between TM0021 and the PII protein was 2.0 Å over 76 aligned residues with 25% sequence identity. TM0021 has one distant homologue in the Thermotoga proteome: TM0403, a PII-like nitrogen regulatory protein (glnK) with 16% sequence identity.7 Based on the structural homology of TM0021 to PII and its sequence homology to TM0403, we conclude that TM0021 and TM0403 are the T. maritima equivalents of glnB2 and glnK,8 respectively. Models for TM0021 homologues can be accessed at http://www1.jcsg.org/cgi-bin/models/get_mor.pl?key=tm0021. The structure reported here represents a PII-like protein in complex with ADP, whose structure has been determined by X-ray crystallography using the SAD method. The information reported here will provide valuable insights into the determinants for signal transduction and thermal stability of this protein family. TM0021 (TIGR: TM0021; Swissprot: Q9WXM9) was amplified by PCR from T. maritima strain MSB8 genomic DNA using PfuTurbo (Stratagene) and primer pairs encoding the predicted 5′- and 3′-ends of TM0021. The polymerase chain reaction (PCR) product was cloned into plasmid pMH1, which encodes a purification tag consisting of the amino acids 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 E.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 was 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 Buffer (20 mM Tris pH 7.9, 150 mM NaCl, 0.25 mM TCEP) and concentrated for crystallization assays to 17 mg/mL by centrifugal ultrafiltration (Millipore). The protein was crystallized using the nanodroplet vapor diffusion method9 with standard JCSG crystallization protocols.1 The crystallization solution contained 2.0 M (NH4)2SO4, 0.2 M NaK-tartrate, and 0.1 M citrate at pH 5.6. The crystals were indexed in the cubic space group P4332 (Table I). Anomalous diffraction data were collected at APS (Chicago, IL) on beamline 19BM at wavelengths corresponding to the inflection point and the low-energy remote of a Selenium MAD experiment (Table I). All data sets were collected at 100 K using an APS-1C charge-coupled device (CCD) detector. Data were integrated and scaled using the HKL2000 program suite.10 Data statistics are summarized in Table I. The structure was determined by using the CCP4 program suite11 and SOLVE / RESOLVE.12 SAD phasing with the data set corresponding to the inflection point (λ1SADSe) gave the best solution. Structure refinement was performed using a merged λ0Se/λ1SADS data set using REFMAC5,11 O,13 and Xfit.14 Refinement statistics are summarized in Table I. The final model includes 1 protein molecule (residues 1–46, 60–100 plus His −1 and 0 from the purification tag), 1 ADP molecule (copurified), 2 sulphates, and 52 water molecules in the asymmetric unit. Residues 47–59 and 101–102 were not visible in the electron density maps. Analysis of the stereochemical quality of the models was accomplished using the JCSG Validation Central suite, which integrates 7 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 TM0021 have been deposited with the PDB and are accessible under the code 1O51. 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).