Crystal structure of a transcription regulator (TM1602) from Thermotoga maritima at 2.3 Å resolution
Year: 2007
Type: article
Abstract: The TM1602 gene of Thermotoga maritima encodes a DNA-binding protein with a molecular weight of 19,423 Da (residues 1–175) and a calculated isoelectric point of 5.8. The TM1602 protein is proposed to contain a 3-histidine (3H) domain [Pfam accession code: PF02829],1 named after its three highly conserved histidines, and a DNA-binding domain. TM1602 represents the first structure of a 3H protein family member. Based on sequence analysis, its function has been postulated to involve binding to small molecules.1 Recent experimental data support this prediction, suggesting that TM1602 belongs to a family of de novo NAD synthesis pathway regulators2 that may bind to nicotinamide, nicotinic acid, or other substrate/products. When activated by these small molecules, which bind to the C-terminal regulatory domain (3H), the N-terminal DNA-binding domain of TM1602 is proposed to bind to the NAD promoter region of the Thermotoga maritima genome and repress the de novo NAD biosynthesis operon. This pathway has been well characterized in an homologous protein, yrxA, from Bacillus subtilis2 and experimental characterization of TM1602 is now in progress [Rodionov and Osterman, personal communication]. Here, we report the crystal structure of TM1602, which was determined using the semi-automated, high-throughput pipeline of the Joint Center for Structural Genomics (JCSG),3 which is part of the National Institute of General Medical Sciences (NIGMS) Protein Structure Initiative (PSI). The TM1602 gene from Thermotoga maritima (TIGR: TM1602, Swissprot: Q9X1T8) was amplified by the polymerase chain reaction (PCR) from genomic DNA using PfuTurbo (Stratagene) and primers corresponding to the predicted 5′- and 3′-ends. The PCR product was cloned into plasmid pMH2T7, which encodes an expression and purification tag (MGSDKIHHHHHH) at the amino terminus of the full-length protein. The cloning junctions were confirmed by DNA sequencing. Protein expression was performed in a selenomethionine-containing medium using the Escherichia coli methionine auxotrophic strain DL41. At the end of fermentation, lysozyme was added to the culture to a final concentration of 1 mg/mL. After a freeze/thaw step, cells were sonicated in lysis buffer [50 mM Tris pH 7.9, 50 mM NaCl, 1 mM MgCl2, 0.25 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP)], and the lysate was clarified by centrifugation at 3400 × g for 60 min. The soluble fraction was applied to nickel-chelating resin (Amersham Biosciences) pre-equilibrated with equilibration buffer [50 mM potassium phosphate pH 7.8, 20 mM imidazole, 300 mM NaCl, 10% (v/v) glycerol, 0.25 mM TCEP]. The resin was washed with wash buffer [50 mM potassium phosphate pH 7.8, 40 mM imidazole, 300 mM NaCl, 10% (v/v) glycerol, 0.25 mM TCEP], and the target protein was eluted with elution buffer [20 mM Tris pH 7.9, 300 mM imidazole, 10% (v/v) glycerol, 0.25 mM TCEP]. The eluate was buffer exchanged with buffer Q [20 mM Tris pH 7.9, 50 mM NaCl, 5% (v/v) glycerol, 0.25 mM TCEP] and loaded onto a RESOURCE Q column (Amersham Biosciences) pre-equilibrated with the same buffer. The flow-through fraction, which contained the target protein, was buffer exchanged with crystallization buffer [20 mM Tris pH 7.9, 150 mM NaCl, 0.25 mM TCEP] and concentrated for crystallization assays to 23 mg/mL by centrifugal ultrafiltration (Millipore). The protein was crystallized using the nanodroplet vapor diffusion method4 with standard JCSG crystallization protocols.3 The crystallization reagent contained 0.8 M NaH2PO4, 0.8 M KH2PO4, and 0.1 M HEPES pH 7.5. Glycerol was added as a cryoprotectant to a final concentration of 25% (v/v). The crystals were indexed in cubic space group P23 (Table I). Molecular weight and oligomeric state of the target protein were determined using a 1 cm × 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 the Stanford Synchrotron Radiation Laboratory (SSRL, Stanford, USA) on beamline BL9-2 at wavelengths corresponding to the high energy remote (λ1), peak (λ2), and inflection (λ3) of a selenium MAD experiment using the BLU-ICE7 data collection environment. The data sets were collected at 100 K using a Q4 CCD detector (ADSC). The MAD data were integrated and reduced using Mosflm8 and then scaled with the program SCALA from the CCP4 suite.5 The structure was determined to 2.3 Å using the CCP4 suite5 and SOLVE/RESOLVE.9 Automatic model building was performed with RESOLVE and iterative ARP/wARP runs.10 Model completion and refinement were performed with the high energy remote data set λ1 using O11 and CNS.12 Data and refinement statistics are summarized in Table I. Analysis of the stereochemical quality of the model was accomplished using AutoDepInputTool,13 MolProbity,14 SFcheck 4.0,15 and WHATIF 5.0.16 Protein quaternary structure analysis was performed using the PQS server.17 Figures were prepared with PyMOL (DeLano Scientific). Atomic coordinates and experimental structure factors for TM1602 from Thermotoga maritima at 2.3 Å resolution have been deposited in the PDB and are accessible under the code 1j5y. The crystal structure of TM1602 (Fig. 1) was determined to 2.3 Å resolution using the MAD method. Data collection, model, and refinement statistics are summarized in Table I. The final model includes a monomer (residues 3–174), one Ni2+ ion, one K+ ion, and 97 water molecules in the asymmetric unit. The expression and purification tag, Met1, His2, and Gly175 were disordered. The Matthews' coefficient (Vm)18 is 2.98 Å3/Da, and the estimated solvent content is 58.5%. The Ramachandran plot, produced by MolProbity,14 shows that 98.8% and 100% of the residues are in favored and allowed regions, respectively. Crystal structure of TM1602. (A) Stereo ribbon diagram colored from the N-terminus (blue) to C-terminus (red). Helices (H1–H7) and β-strands (β1–β6) are labeled. (B) Diagram showing the secondary structural elements superimposed on the primary sequence. The helices, β-strands, β-turns, and γ-turn are indicated. β-hairpins are depicted by a red loop. Dashed lines indicate that no electron density was observed for that region. TM1602 is composed of six β-strands (β1–β6), six α-helices (H1–H3, H4, H5, and H7), and two 310-helices (H4′ and H6) [Fig. 1(A,B)]. The total β-sheet, α-helical, and 310-helical content is 23.3, 43.6, and 2.9%, respectively. The TM1602 monomer is comprised of two structural domains, an N-terminal DNA-binding domain (residues 3–67) and a C-terminal 3H domain (residues 68–174), that, surprisingly, adopts an HPr-like fold. The DNA-binding domain consists of three α-helices (H1–H3) and one small β-hairpin (β1 and β2), which form a typical “winged helix” DNA-binding motif. The histidine-containing phosphocarrier (HPr)-like fold of the 3H domain is characterized by two layers, a helical layer (H7, H6, H4′, H4, and H5) and a twisted anti-parallel β-sheet layer (β3, β6, β4, and β5), with a 1423 topology. The order of secondary structural elements in the 3H domain is βαββαβα, where the α-helices are packed on one side of the four-stranded β-sheet, a topology that is conserved among all HPr proteins. Embedded between the layers, the three highly conserved histidines (His79, His146, and His148) and a glutamate (Glu87) form a metal binding site. These residues are conserved in both archaeal and bacterial homologs. At the entrance of the binding site, a fourth histidine (His105), conserved only in bacteria, is located between strands β4 and β5, and one can speculate that it regulates access to the binding site by modulating interactions between two entrance loops (H5 to β6 and β4 to β5). A transition metal ion and a water molecule occupy the metal binding site in the 3H domain of TM1602 (Fig. 2B). However, the available data do not allow unambiguous assignment of the metal in the structure. Based on coordination geometry, anomalous difference Fourier peaks, refinement results, and similar ligating residues in the MDB,19 Ni2+, Cu2+, or Zn2+ are the most probable metals in the structure. Since the protein was exposed to Ni2+ during purification, Ni2+ was included in the final model. Coordination of the metal ion is by three histidines (His79, His146, and His148), a glutamate (Glu87), and a water molecule with distances of 2.38, 2.30, 2.12, 2.10, and 2.31 Å, respectively. Comparisons of TM1602 to proteins that bind Ni2+ ions under physiological conditions19 do not reveal any structural similarity in the binding site. However, some similarity is found to Zn2+-binding sites, although the geometry of metal binding is different. Structural comparison of TM1602. (A) Structure alignment of the 3H domain of TM1602 (residues 68–174; grey) and histidine-containing phosphocarrier protein HPr from Streptococcus faecalis (PDB accession code: 1ptf; blue). The Ni2+ ligand (magenta) and the water molecule (green) from TM1602 are shown. (B) TM1602 active site. Conserved active site histidines (His79, His146, and His148) and “gating” histidine (His105) are shown in ball-and-stick representation. The Ni2+ ion (magenta) and water molecule (green) are shown at the active site. (C) Crystallographic packing of TM1602 in its tetrameric form, as defined by the PQS server.17 Each monomer is shown in a different color. (D) TM1602 in its dimeric form that is proposed as the possible dsDNA-binding form. Arrows represent proposed a ligand-induced conformational of change that could enable binding to dsDNA. A structural similarity search, performed with the coordinates of the 3H domain of TM1602 using the DALI server,20 finds the histidine-containing phosphocarrier protein, HPr, from Streptococcus faecalis (PDB: 1ptf) among the top hits. The RMSD between TM1602 and S. faecalis HPr is 2.7 Å over 87 aligned residues (Cα atoms) with 17% sequence identity. The DALI structural alignment reveals differences in the loop region between helices H5 and H6 and the loop between β-sheets β4 and β5; in TM1602, these loops extend much further (Fig. 2A). HPr is a small phosphocarrier protein that is part of the sugar transport system called the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The PTS consists of a series of proteins and enzymes involved in the transfer of phosphate from phosphoenolpyruvate to sugar to form sugar phosphate. The consensus description of the binding site for HPr proteins involves a highly conserved histidine (His15) that undergoes a conformational change upon phosphorylation to allow the phosphoryl group to form a hydrogen bond with the main-chain amide nitrogen of residue 16 and a conserved arginine (Arg17).21 Structural alignments of TM1602 with S. faecalis HPr and the HPr protein from E. coli (PDB: 1hdn) show that the active site residues corresponding to HPr activity are not conserved. Moreover, TM1602 contains a DNA-binding domain that is not found in HPr proteins. Although the 3H domain of TM1602 adopts an HPr-like fold, it is unlikely that it participates in PTS, or has an HPr-like function, given that key active site residues are not conserved and it contains a DNA-binding domain. A PSI-BLAST22 search using TM1602 revealed that homologs exist in both archaea and bacteria, but not in eukaryotes. In bacteria, the 3H domain is paired with the N-terminal DNA-binding domain, suggesting that these proteins are perhaps involved in a DNA-related process. In contrast, the 3H domains in thermophilic archaeal proteins are coupled with a TrkA domain, which has been associated with phosphate23, 24 and NAD+25 binding. Multiple sequence alignment of the 3H domain shows conservation of the three binding site histidines in all homologs. In all bacterial homologs, including Thermotoga, the “gating” histidine (His105) and an active site glutamate residue (Glu87) are additionally conserved, whereas in archaea they are not, suggesting that these residues play a regulatory role only in bacterial proteins. Through comparative genomic analysis, TM1602 has been identified to belong to a family of de novo NAD synthesis pathway regulators. Biosynthesis of NAD and NADPH is regulated on the level of transcription by repressor proteins: NadR in enterobacteria26-29 and YrxA in Bacillus subtilis.2 Analysis of the B. subtilis genome identified a novel nicotinate-responsive transcriptional regulator encoded by the proximal gene yrxA that represses the de novo NAD biosynthesis operon. YrxA was found to bind specifically to an NAD promoter region in the presence of nicotinic acid. Phylogenetic footprinting analysis conducted by Rodionov and Osterman (personal communication) identified a conserved YrxA-binding signal in upstream regions of NAD biosynthesis operons of members from Bacillus/Clostridium groups and in T. maritima (TM1602). Multiple sequence alignment of YrxA proteins show conservation of both the DNA-binding domain and the 3H domain, specifically the three histidine residues, further supporting their putative regulatory function. Analysis of the crystallographic packing of TM1602 using the PQS server17 indicates a tetramer (dimer of dimers) (Fig. 2C). However, a dimer rather than the tetramer is more likely to represent the dsDNA-binding form. In the tetramer, steric hindrance from one of the dimers would prevent DNA binding. A dimer is also consistent with results from analytical size exclusion chromatography in combination with static light scattering. Interestingly, the distance between the DNA-binding domains of the homodimer exceeds that between the adjacent major grooves of dsDNA. Therefore, the N-terminal domains of the homodimer (Fig. 2D) would have to undergo a ligand-induced, domain motion in order to optimally bind dsDNA. Similar mechanisms of regulation have been noted in other transcriptional regulators, such as the tetracycline repressor protein (TetR). The binding of an inducer molecule (tetracycline) in TetR induces conformational change and domain motion of the DNA-binding region, causing release from dsDNA.30 We propose a similar mechanism for TM1602 whereby ligand-binding at the 3H domain (regulatory region) causes the DNA-binding domains to move closer to each other, thus enabling them to bind dsDNA. Whether the tetramer observed in our structure has any biological relevance and represents an additional level of regulation or is simply an artifact of crystallization is unclear at this time. Another significant DALI hit was to a putative glycine cleavage system transcriptional repressor from Vibrio cholerae (PDB: 1u8s), which belongs to a ferredoxin-like fold, with an RMSD of 2.2 Å over 171 aligned residues (Cα atoms) and 15% sequence identity. Recently, Vesterstrom and Taylor31 presented a method of flexible structural alignment to compare protein structures. They applied this method to identify existing and putative circular permutations in proteins. Specifically, they identified the HPr-like domain of TM1602 was similar to a periplasmic divalent cation tolerance protein from Archaeoglobus fulgidus (PDB: 1p1l), which has a ferredoxin-like fold. Furthermore, they reported that the ferredoxin-like fold differs from the HPr-like fold in that it contains a large insert and a circular permutation. Although the 3H domain of TM1602 shares structural similarity with a ferredoxin-like fold, it is unlikely that they share similar functionality. The TM1602 protein family contains several homologous sequences mostly of bacterial origin. Models for TM1602 homologs can be accessed at http://www1.jcsg.org/cgi-bin/models/get_mor.pl?key=1j5y. The information presented here, in combination with further biochemical and biophysical studies, should yield valuable insights into the functional role and the evolutionary history of this important regulatory protein. Portions of this research were carried out at the Stanford Synchrotron Radiation Laboratory (SSRL). 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).
DOI: 10.1002/prot.21221
Authors: Dana Weekes, Mitchell D. Miller, S. Sri Krishna, Daniel McMullan, Timothy M. McPhillips, Claire Acosta, Jaume M. Canaves, Marc‐André Elsliger, Ross Floyd, Slawomir K. Grzechnik, Lukasz Jaroszewski, Heath E. Klock, Eric Koesema, John S. Kovarik, Andreas Kreusch, Andrew T. Morse, Kevin Quijano, Glen Spraggon, Henry van den Bedem, Guenter Wolf, Keith O. Hodgson, John Wooley, Ashley M. Deacon, Adam Godzik, Scott A. Lesley, Ian A. Wilson
Language: English
Subfield: Materials Chemistry
Field: Materials Science
Domain: Physical Sciences
Open Access status: bronze
OpenAlex ID: W2135319108