Crystal structure of THEP1 from the hyperthermophile Aquifex aeolicus: a variation of the RecA fold
- Michael Roßbach†1,
- Oliver Daumke†2,
- Claudia Klinger1,
- Alfred Wittinghofer2 and
- Michael Kaufmann1Email author
© Roßbach et al; licensee BioMed Central Ltd. 2005
Received: 18 December 2004
Accepted: 20 March 2005
Published: 20 March 2005
aaTHEP1, the gene product of aq_1292 from Aquifex aeolicus, shows sequence homology to proteins from most thermophiles, hyperthermophiles, and higher organisms such as man, mouse, and fly. In contrast, there are almost no homologous proteins in mesophilic unicellular microorganisms. aaTHEP1 is a thermophilic enzyme exhibiting both ATPase and GTPase activity in vitro. Although annotated as a nucleotide kinase, such an activity could not be confirmed for aaTHEP1 experimentally and the in vivo function of aaTHEP1 is still unknown.
Here we report the crystal structure of selenomethionine substituted nucleotide-free aaTHEP1 at 1.4 Å resolution using a multiple anomalous dispersion phasing protocol. The protein is composed of a single domain that belongs to the family of 3-layer (α/β/α)-structures consisting of nine central strands flanked by six helices. The closest structural homologue as determined by DALI is the RecA family. In contrast to the latter proteins, aaTHEP1 possesses an extension of the β-sheet consisting of four additional β-strands.
We conclude that the structure of aaTHEP1 represents a variation of the RecA fold. Although the catalytic function of aaTHEP1 remains unclear, structural details indicate that it does not belong to the group of GTPases, kinases or adenosyltransferases. A mainly positive electrostatic surface indicates that aaTHEP1 might be a DNA/RNA modifying enzyme. The resolved structure of aaTHEP1 can serve as paradigm for the complete THEP1 family.
Comparative genomics led to the definition of 4873 clusters of orthologous groups of proteins (COGs) by comparing protein sequences encoded in (currently 66) completely sequenced genomes . Aimed at finding thermophile-specific proteins among bacteria, extended phylogenetic patterns searches based on the COG-database were performed. Using this strategy, COG1618 was detected as a cluster containing proteins from all thermophilic and hyperthermophilic but only one mesophilic organism [2–4]. Surprisingly, although also absent from unicellular eukaryotes, COG1618-homologs are present in many higher multicellular organism such as Homo sapiens, Mus musculus, Danio rerio, Rattus norvegicus, etc. Because of this unusual phylogenetic distribution, aaTHEP1, the gene product of aq_1292 from the hyperthermophilic bacterium Aquifex aeolicus, was characterised biochemically as the first member of COG1618 proteins . The analysis revealed that aaTHEP1 is an NTPase catalyzing ATP and GTP hydrolysis at turnover rates of 5 × 10-3 s-1and 9 × 10-3 s-1, respectively, with a Km in the micromolar range and a temperature optimum between 70 and 80°C. Although COG1618 proteins are annotated as "predicted nucleotide kinases"such an activity could not be confirmed for aaTHEP1 experimentally and its in vivo function remains unknown. To further characterize the aaTHEP1 function, we resolved its three dimensional structure by X-ray crystallography.
Results and discussion
Overall structure, domain class and architecture
Data collection and refinement statistics A summary of all relevant crystallographic parameters during data collection and the refinement procedures is shown.
Swiss Light Source X06SA
a = 35.0 Å b = 64.2 Å c = 39.6 Å
α = 90.0° β = 105.2° γ = 90.0°
2.17 Å 3/Da
λpeak = 0.97625 Å
λinfl = 0.97980 Å
20 - 1.4 Å
20 - 1.4 Å
R symm a,b total
R symm a,b last shell
I/σ(I) last shell
Anomalous phasing power
λinfl = 1.7 λpeak = 1.8
Anomalous phasing power last shell
λinfl = 0.46 λpeak = 0.48
FOM last shell
FOM after solvent flattening
FOM after solvent flattening, last shell
18.2 - 1.4 Å
Reflections unique (test set)
Number of amino acids
Number of atoms
Number of water molecules
In summary, the overall topology of aaTHEP1 is a central sheet with helical structures on each side. According to the CATH protein structure classification , aaTHEP1 is assigned to class 126.96.36.1990 i. e. "P-loop containing nucleotide triphosphate hydrolases, homologous superfamilies with Rossmann fold topology" which are mixed alpha-beta proteins with 3-layer(α/β/α) sandwich architecture.
Structural alignments and fold classification
For comparison with other structures in the pdb-database, the DALI algorithm was employed . The closest homologue of aaTHEP1 was found to be cob(I)alamin adenosyltransferase (pdb-code: 1G5R, Z-score = 9.9) that catalyzes the final step in the conversion of vitamin B(12) to coenzyme B(12) and has a RecA-like protein fold. A comparison between the topologies of aaTHEP1, cob(I)alamin adenosyltransferase and RecA clearly shows the structural similarity (Fig. 3) despite only 9% sequence identity in the aligned region. In contrast to cob(I)alamin adenosyltransferase and RecA, aaTHEP1 contains an extension of its β-sheet consisting of strands β3-β6. We conclude that the structure of aaTHEP1 represents a variation of the RecA protein fold.
Topology of the P-loop
The catalytic centre
No electron density for an ADP molecule was found indicating that only the nucleotide-free protein crystallized. However, we found electron density for a phosphate ion in the putative nucleotide binding site where the β-phosphate of the nucleotide is expected. This is a usual phenomenon, since negatively charged ions are often found in empty nucleotide binding sites (e. g. ).
In other ATPases and GTPases, the aspartate residue of the consensus site DxxG (D106 in aaTHEP1) is involved in positioning a water-bridged magnesium ion presumably important for nucleotide hydrolysis [10, 11]. In the nucleotide free aaTHEP1, there is also a magnesium ion at the corresponding position which is octahedrally coordinated to the hydroxyl group of T14 of the P-loop, a phosphate oxygen and four water molecules. One of these water molecules (W24) makes a hydrogen bond to D106. Thus, the arrangement of the magnesium ion is similar as this found in the nucleotide-bound conformation of other ATPases and GTPases.
The protein surface
The location of conserved residues in a protein structure often points to sites which are functionally important, e. g. the catalytic centre or conserved binding sites . To detect putative binding sites of aaTHEP1, we colour coded the surface of aaTHEP1 with respect to the conservation of exposed amino acids. As can be seen in Figure 5, there is only one highly conserved region located in and around a cleft of the protein surface which includes the Walker A motif (P-loop). We conclude that this particular region represents the functionally most important site, i. e. the nucleotide and cosubstrate binding site of aaTHEP1. Not even a single amino acid residue conserved in all species aligned in Figure 1 can be detected on the residual protein surface of aaTHEP1. For that reason, we conclude that binding of the physiological cosubstrate is restricted to the neighbourhood of the nucleotide binding pocket.
Analysis of the electrostatic surface potential of aaTHEP1 strikingly revealed a number of positively charged clusters, whereas almost no negatively charged regions can be found (Figure 5). This is in agreement with the strong binding of aaTHEP1 to cation exchangers and its theoretical pI of 9.9. The largest positively charged spot is located in a conserved region close to the nucleotide binding cleft. Based on this observation and the similarity to the RecA protein we speculate that aaTHEP1 may be a DNA or RNA modifying enzyme. Gene functions can be predicted by searching for the conservation of operons and gene orders because genes found in gene strings, particularly in multiple genomes, can be assumed to be functionally linked . For THEP1, we detected 4 genomes (Aeropyrum pernix K1, Archaeoglobus fulgidus DSM 4304, Thermoplasma acidophilum DSM 1728 and Thermoplasma volcanium GSS1) where the THEP1-gene is immediately followed by a COG1867 protein on the same strand. In Pyrococcus furiosus, this protein is characterized as a N2, N2-dimethylguanosine tRNA methyltransferase . Thus, aaTHEP1 may also play a role in tRNA modification. Furthermore, both COG1867 proteins and THEP1 proteins can be considered to belong to the group of PACE-proteins (proteins from Archaea without assigned function that are conserved in Eukarya) . PACE proteins are described being involved in fundamental cellular functions and several of them are obviously related to RNA metabolism .
The human homologue
The human homologue MGC13186 (hsTHEP1) shows 39% sequence identity to aaTHEP1 (Figure 1) and was first described in a study aiming at identifying full-length ORF for all human and mouse genes. No function is yet described for this protein. However, gene profiling data from UniGene are available . hsTHEP1 is widely expressed in most of the examined tissues including brain, heart, lymph node, skin and pancreas whereas no expression was found in blood, thymus, bladder and spleen. It is especially highly expressed in embryonic and various tumour tissues. From these data we conclude that hsTHEP1 has a general function in many human tissues.
The crystal structure of aaTHEP1 uncovered a modified RecA-like fold. Although the function of aaTHEP1 remains unclear, the structure led us to conclude that the enzyme does not belong to the group of GTPase, kinases or adenosyltransferases. Analysis of the electrostatic surface potential revealed several positively charged clusters indicating the presence of putative nucleic acid binding sites. Since aaTHEP1 has homologues in thermophilic bacteria and vertebrates it can serve as a model for the complete COG1618 protein family.
To aid a consistent nomenclature of the THEP1 protein family we propose to adopt the name THEP1 to all members across the species, e.g. hsTHEP1 for the human protein, mmTHEP1 for the mouse protein, etc..
Crystallization, data collection, processing, structure solution, refinement and validation
Recombinant aaTHEP1 was purified from Escherichia coli as described earlier . Bacteria were grown in minimal media without methionine containing 50 mg/l L-selenomethionine . Crystals of the dimension 250 × 80 × 35 μm3 were obtained by the hanging drop method after mixing equal volumes of 13 mg/ml aaTHEP1 with reservoir buffer containing 15 % PEG-3350 and 0.1 M potassiumdihydrogenphosphate. For cryo-protection, crystals were soaked for 10 sec in 30 % PEG-3350, 200 mM potassiumdihydrogenphosphate and flash-frozen in liquid nitrogen. The diffraction data were collected at the Swiss Light Source (SLS) from a single crystal. Data were processed and scaled using XDS  and XSCALE . The positions of the three selenium sites in the asymmetric unit were determined using SHELXD . Those positions were refined and the electron density of the protein calculated by SHARP . Solvent flattening and histogram matching were done by SOLOMON  and DM . ARP/WARP was used to automatically build 85% of the backbone and sidechains . For further model interpretation XFIT XtalView  was used. Refinements were made with Refmac . PROCHECK  and Whatcheck  were used to validate the structure. Secondary structures were calculated using DSSP [32, 33]. DALI-searches  were carried out at , GRATH  at  and further structural comparisons using SSAP  were done at . BLAST was performed at . Figure 1 was prepared using GeneDoc available at . All figures depicting structures were prepared using PyMol  or Swiss pdb-viewer [42, 43]. The X-Ray coordinates and structure factors have been deposited in the PDB database under pdb-code 1YE8.
We are grateful to the machine and beamline groups whose outstanding efforts have made these experiments possible. We would like to thank Dr. John Doe for his support in setting up the beamline and Dr. Karin Muller for her help in analyzing our data. We also wish to thank Dr. Ilme Schlichting for collecting the data at the SLS, Drs Michael Weyand and Ingrid Vetter for their help in data analysis and Astrid Böhm for carrying out the fermentations.
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