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PROTEIN STRUCTURE REPORT

NMR structure of the conserved hypothetical protein TM0487 from Thermotoga maritima: Implications for 216 homologous DUF59 proteins

The Scripps Research Institute (TSRI), Department of Molecular Biology and Joint Center for Structural Genomics, La Jolla, California 92037, USA

Reprint requests to: Kurt Wüthrich, The Scripps Research Institute, Department of Molecular Biology and Joint Center for Structural Genomics, La Jolla, CA 92037, USA; e-mail: wuthrich{at}scripps.edu; fax: (858) 784-8014.

(RECEIVED August 5, 2005; FINAL REVISION August 5, 2005; ACCEPTED August 10, 2005)

	Abstract

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The NMR structure of the conserved hypothetical protein TM0487 from Thermotoga maritima represents an /-topology formed by the regular secondary structures 1–1–2–2–3–4–3– 5–3₁₀–4, with a small anti-parallel -sheet of -strands 1 and 2, and a mixed parallel/anti-parallel -sheet of -strands 3–5. Similar folds have previously been observed in other proteins, with amino acid sequence identity as low as 3% and a variety of different functions. There are also 216 sequence homologs of TM0487, which all have the signature sequence of domains of unknown function 59 (DUF59), for which no three-dimensional structures have as yet been reported. The TM0487 structure thus presents a platform for homology modeling of this large group of DUF59 proteins. Conserved among most of the DUF59s are 13 hydrophobic residues, which are clustered in the core of TM0487. A putative active site of TM0487 consisting of residues D20, E22, L23, T51, T52, and C55 is conserved in 98 of the 216 DUF59 sequences. Asp20 is buried within the proposed active site without any compensating positive charge, which suggests that its pK_a value may be perturbed. Furthermore, the DUF59 family includes ORFs that are part of a conserved chromosomal group of proteins predicted to be involved in Fe–S cluster metabolism.

Keywords: NMR structure determination; Thermotoga maritima; structural genomics; DUF59 proteins

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051755805.

	Introduction

TOP Abstract Introduction Results Discussion Materials and methods References

 
Proteins containing a domain of unknown function 59 (DUF59) have been found in bacteria, archaea, and some eukaryotes (COG5133). There are reports that some of these proteins are involved in the degradation of phenylacetic acid (Ferrández et al. 1998; Olivera et al. 1998) and in metal-sulfur cluster synthesis (Lezhneva et al. 2004; Stöckel and Oelmüller 2004). No three-dimensional structure of a DUF59 has been reported yet, and for many of these proteins, no function has been identified.

The Thermotoga maritima protein TM0487 (Nelson et al. 1999) contains the signature sequence of the DUF59 family. This 103-residue conserved hypothetical protein was selected for structural studies in the context of the research program of the Joint Center for Structural Genomics (JCSG) (Lesley et al. 2002). TM0487 has a molecular weight of 11,490 Da and a calculated isoelectric point of 4.39 and was identified as a target for NMR structure determination based on 1D ¹H NMR screening (Peti et al. 2004). This study describes the NMR structure determination of TM0487, and based on three-dimensional structure comparisons and amino acid sequence analysis, we discuss structural and functional roles of the conserved residues found in proteins belonging to the DUF59 family.

	Results

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NMR structure determination
TM0487 was expressed in Escherichia coli and purified as described previously (Almeida et al. 2004). The three-dimensional structure of TM0487 in solution has been determined using standard heteronuclear NMR methods with the uniformly ¹³C/¹⁵N-labeled protein for the sequence-specific backbone and side-chain assignments (Wüthrich 1986; Sattler et al. 1999). The ATNOS/CANDID/DYANA software package (Güntert et al.1997; Herrmann et al. 2002a,b) was used for combined automatic NOESY peak picking, NOE assignment, and three-dimensional structure calculation. Experimental details are given in Materials and Methods. The parameters for the NMR structure determination of TM0487 in Table 1 show that a high-quality structure was obtained, with above-average local disorder limited to the N-terminal tetrapeptide and C-terminal hexapeptide segments (Fig. 1A).

View larger version (49K): [in this window] [in a new window]   Figure 1. Three-dimensional structure of TM0487. (A) Stereo view of a bundle of 20 energy-minimized DYANA conformers of TM0487. The polypeptide backbone is shown as a gray spline function through the C-positions. The side chains are drawn as all-heavy-atom structures. Selected residue positions are identified by the sequence numbers. The side chains of residues with average local displacements of <1.0Å are shown in blue, and those of the less well-ordered residues are in red. (B) Ribbon presentation of the closest conformer to the mean coordinates of the bundle of 20 conformers used to represent the NMR structure. The chain termini P1 and V103, as well as the regular secondary structures are identified.

	Discussion

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Sequence homology between TM0487 and DUF59 proteins
A search for conserved domains using the RPS-BLAST algorithm (Marchler-Bauer et al. 2002) with the cdd. v1.65 database revealed a high amino acid sequence identity of TM0487 with the DUF59s (Fig. 2A). The DUF59s are mainly characterized by conserved hydrophobicity at several specific sequence positions, an acidic residue corresponding to position 20 in TM0487, and a cysteine at position 55, where the latter two amino acids are strictly conserved among 216 nonredundant sequences with high similarity to TM0487 (E<0.01). The DUF59 family includes PaaD proteins (Ferrández et al. 1998; Olivera et al. 1998), which show about 25% sequence identity with TM0487. The PaaD proteins are presumed to be part of the phenylacetate degradation pathway. This pathway has not been identified as yet in T. maritima, and in particular, the pathway "signature enzyme," phenylacetate-CoA ligase (EC 6.2.1.30 [EC] ) has not been found. Absence of this aerobic pathway in T. maritima would not be a surprise, since it is not really expected to be present in anaerobes. Other closely homologous proteins to TM0487 are part of a conserved chromosomal cluster of proteins related to sulfur metabolism, including cysteine desulfurase, IscU, and IscA (HESB)-like proteins involved in Fe–S cluster biosynthesis, and ABC-type transport systems related to Fe–S cluster assembly (Zheng et al. 1993, 1998). Overall, proteins containing a DUF59 domain cover a wide range of different physiological functions, and with this availability of the three-dimensional structure of TM0487 they can now be modeled by standard homology modeling tools, such as SWISS-MODEL (Guex and Peitsch 1997).

View larger version (68K): [in this window] [in a new window]   Figure 2. Sequence and three-dimensional structure analysis in the DUF59 family. (A) Alignment of the 21 most diverse amino acid sequences of the 98 DUF59 domains that contain a local structure resembling the putative active site in TM0487 (see text). The bars and the arrows at the top indicate the locations of -helices and -strands, respectively, in the structure of TM0487, and the residue numbering of TM0487 is given above its sequence. The sequence positions with strict conservation of hydrophobic amino acids located in the core of the TM0487 structure are shown in brown, and those suggested in the text to be part of the active site of TM0487 are shown in green. Common features of the 21 proteins are identified at the bottom by the amino acid one-letter code for strictly conserved amino acids, the symbol # for hydrophobic residues, the symbol @ for acidic residues, and the symbol for Thr or Ser. The sequences are identified by the GI NCBI accession numbers. (B) The polypeptide backbone of the conformer with the lowest RMSD to the mean coordinates of the bundle of 20 energy-minimized conformers in Figure 1A is represented by a gray spline function through the C-positions. The brown side chains identified by brown numerals represent those hydrophobic residues in TM0487 that have conserved hydrophobicity in the 216 DUF59s, and are proposed in the text to be key factors for stabilization of the DUF59 fold. The green residues are highly conserved in the sequence alignment of Figure 2A, and are all located within 5.0 Å of at least one other green residue. In the text, we propose that these residues form a putative active site of TM0487 (see also Fig. 3).

Identification of a putative active site
Thirteen hydrophobic residues conserved in the DUF59s are positioned in the core of the TM0487 structure (shown in brown in Fig. 2B), suggesting that they are involved in the stabilization of closely related folds in all of these proteins. An acidic residue at position 20 and the cysteine at position 55 are proximal to each other in the structure of TM0487 (in the bundle of conformers of Fig. 1A, the average distance between C of D20 and S of C55 is 6.3 Å). A search for similar three-dimensional structure patterns in nonhomologous tertiary structures, using the PINTS server (Stark and Russell 2003), identified closest similarity of TM0487 with the hybrid cluster protein (HCP) from Desulfovibrio desulfuricans (PDB ID 1oa0 [PDB] ), which has not yet been assigned a specific function (Aragão et al. 2003). The 216 nonredundant sequences that contain the DUF59 domain were then realigned by CLUSTALW (1.82; Chenna et al. 2003) in such a way that only sequences with the following traits were retained, i.e., an acidic residue at position 20, T51, T52, or S52, and C55 (residue numbering of TM0487). A serine rather than a threonine at position 52 was found in 37 of these sequences, and was included due to its similarity to threonine. Two gaps were allowed in the sequence alignment (Fig. 2A), to avoid interruption of putative secondary structure elements observed in the TM0487 NMR structure. This alignment revealed three new positions with highly conserved traits, i.e., an acidic residue at position 22, a hydrophobic residue at position 23, and proline at position 56. Among the 216 DUF59s, we thus found a group of 98 nonredundant sequences containing acidic residues at positions 20 and 22; a hydrophobic residue at position 23; threonine 51; threonine or serine at position 52; cysteine 55; and proline 56. Residues 22, 23, 51, 52, and 55 form a contiguous surface in the TM0487 structure (green in Fig. 2B), suggesting that they might form an active site for this class of proteins. It is also worth noting that the buried side-chain carboxyl group of D20 does not have a nearby positively charged group within the protein structure, which may then suggest it has a perturbed pK_a-value. Finally, the strictly conserved residues C55 and P56 (Fig. 2A) are the first two residues in a type IV -turn (Richardson 1981), and proline in position 56 might have a role in properly positioning C55 in the active site.

In a comparison of the putative active site of TM0487 with the structure of one of the Fe–S clusters in the HCP protein from D. desulfuricans, a positive match was identified between the residues T51, T52, and C55 of TM0487, and the residues T75, T12, and C18 in this Fe–S cluster (red labels in Fig. 3). Although there is apparent near-identity in the spatial arrangement of this group of three chemically identical residues (Fig. 3), the three cysteines in positions 6, 9, and 24 of the Fe–S cluster of HCP are missing in TM0487. This does not a priori exclude the presence of an Fe–S cluster in TM0487, since other Fe–S clusters have been described with only two or three cysteines, with the other "cysteine positions" being occupied by Asp or Glu (Aragão et al. 2003; Johnson et al. 2005). In conclusion, although some closely homologous proteins to TM0487 are part of a conserved chromosomal cluster of proteins related to Fe–S cluster assembly (Zheng et al. 1993, 1998), the evidence presently available leaves open whether or not the proposed active site residues (green in Figs. 2B, 3A) might form a novel type of Fe–S cluster environment.

View larger version (48K): [in this window] [in a new window]   Figure 3. (A) Top view of TM0487 derived from Figure 1B by a rotation of 90° about a horizontal axis. The backbone is colored in gray, and the regular secondary structures are identified. The side chains of the residues suggested to be part of the putative active site, as discussed in the text, are shown as green ball-and-stick representations, with the oxygen and sulfur atoms colored in red and yellow, respectively. (B) The Fe–S cluster in the protein HPC from Desulfovibrio desulfuricans (PDB ID 1oa0 [PDB] ) oriented for optimal fit of the residues T75, T12, and C18 with the amino acid side chains T51, T52, and C55 of TM0487 in A (red lettering in both structures). The close similarity between the three-dimensional arrangements of these three-residue clusters in the two proteins was discovered by a PINTS search; in addition, one notes in the resulting orientation of the two structures that the spatial arrangement of the residues C24 and V23 in HPC matches that of the residues D20 and L23 in TM0487. The Fe–S cluster in HPC is identified by big yellow spheres representing the sulfur atoms; the iron atoms have been omitted for improved clarity.

	Materials and methods

TOP Abstract Introduction Results Discussion Materials and methods References

Data collection
All NMR experiments were performed at 313 K on Bruker Avance600 and Avance900 spectrometers equipped with TXI-HCN-z or TXI-HCN-xyz gradient probeheads.

NMR data analysis and structure calculation
The sequence-specific resonance assignments were previously described (Almeida et al. 2004). The ¹H, ¹³C, and ¹⁵N chemical shifts have been deposited in the BioMagResBank (http://www.bmrb.wisc.edu) under accession number 5976.

To collect the input for the structure calculation, three-dimensional ¹⁵N-resolved [¹H,¹H]-NOESY and three-dimensional ¹³C-resolved [¹H,¹H]-NOESY spectra (both recorded at 900 MHz with a mixing time of 75 msec) were processed with PROSA (Güntert et al. 1992) and analyzed with an automated routine for NOESY peak picking and NOE assignment, ATNOS/CANDID (Herrmann et al. 2002a,b) implemented in the torsion angle dynamics program DYANA (Güntert et al. 1997). The input for ATNOS/CANDID/DYANA (Herrmann et al. 2002a, b) consisted of the amino acid sequence of TM0487, the chemical-shift lists obtained from the previous sequence-specific resonance assignment (Almeida et al. 2004), and the three-dimensional heteronuclear-resolved [¹H,¹H]-NOESY spectra. Stereospecific assignments of 42 valine and leucine isopropyl methyl groups had been determined experimentally by biosynthetic fractional ¹³C-labeling (Senn et al. 1989). These 42 stereospecific assignments, and 106 backbone dihedral angle constraints derived from the C and C chemical shifts (Spera and Bax 1991; Luginbühl et al. 1995) were added to the input for each cycle of structure calculation. The standard iterative protocol with seven cycles of ATNOS peak picking, CANDID NOE assignment, and three-dimensional structure calculation with DYANA was applied (Herrmann et al. 2002a,b). During the first six ATNOS/CANDID cycles, ambiguous distance constraints were used (Nilges et al. 1997). In the second and subsequent cycles, the intermediate three-dimensional protein structures were used as an additional guide for the interpretation of the NOESY spectra. For the final structure calculation in cycle 7, only distance constraints were retained that could be unambiguously assigned based on the intermediate protein structure from cycle 6. The 20 conformers from cycle 7 with the lowest residual DYANA target function values were energy minimized in a water shell with the program OPALp (Luginbühl et al. 1996, Koradi et al. 2000), using the AMBER force field (Cornell et al. 1995). The program MOLMOL (Koradi et al. 1996) was used to analyze the resulting bundle of 20 energy-minimized conformers and to prepare the figures showing molecular models.

Data validation and deposition
Analysis of the stereochemical quality of the NMR structure bundle was accomplished using the JCSG Validation Central Suite (http://www.jcsg.org), which integrates seven validation tools: Procheck 3.5.4, Sfcheck 4.0, Prove 2.5.1, ERRAT, WASP, DDQ 2.0, and Whatcheck. The atomic coordinates of the refined structure have been deposited in the PDB (http://www.rcsb.org/pdb), with codes 1uwx [PDB] for the bundle of 20 structures and 1wcj for the structure with the smallest RMSD to the mean coordinates of the bundle.

	Footnotes

 
¹ Present addresses: Institute for Molecular Biology and Biophysics, Eidgenössische Technische Hochschule Zürich, CH-8093 Zürich, Switzerland

² Department of Molecular Pharmacology, Physiology and Biotechnology, Brown University, Providence, RI 02912, USA.

	Acknowledgments

 
We thank Drs. S.A. Lesley and H.E. Klock for providing us with the genomic materials, and Drs. S.K. Grzechnik and O. Zagnitko for data from the TM bioinformatics analysis. M.S.A. is supported by the Pew Latin American Fellows Program in the Biological Sciences; W.P. was supported by an E. Schrödinger Fellowship (J2145); K.W. is the Cecil H. and Ida M. Green Professor of Structural Biology at TSRI. The use of the high-performance computing facility at TSRI is gratefully acknowledged. Grant sponsor: National Institutes of Health, Protein Structure Initiative; grant no. P50 GM62411, U54 GM074898.

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