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fold
1 NMR Group, Centro Nacional de Investigaciones Oncológicas, 28029 Madrid, Spain
2 Division of Molecular and Structural Biology, Ontario Cancer Institute, and Department of Medical Physics, University of Toronto, Toronto, Ontario, Canada M5G 2M9
3 Unidad de Bioinformática, Centro de Biología Molecular, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain
4 Centro Nacional de Biotecnología, Consejo Superior Investigaciones Científicas (CSIC), Cantoblanco, 28049 Madrid, Spain
5 Instituto de Química-Física "Rocasolano," Consejo Superior Investigaciones Científicas (CSIC), 28006 Madrid, Spain
Reprint requests to: Manuel Rico, Instituto de Química-Física "Rocasolano," CSIC, Serrano 119, 28006 Madrid, Spain; e-mail: mrico{at}iqfr.csic.es; fax: 34-91-5642431.
(RECEIVED January 9, 2004; FINAL REVISION March 3, 2004; ACCEPTED March 3, 2004)
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Abstract |
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 TOP Abstract IntroductionResultsDiscussionMaterials and methodsReferences |
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+
fold, consisting of two -helices (one N terminal and one C terminal) packed on the same side of a central -hairpin. This structure is likely shared by its three orthologs, detected in three other Archaebacteria. There are no clear features in the sequences of these proteins or in the genome organization of Mth to make a reliable functional assignment to this protein. However, the structural similarity to Escherichia coli MinE, the protein which controls that division occurs at the midcell site, lends support to the proposal that Mth677 might be, in Mth, the counterpart of the topological specificity domain of MinE in E. coli.
Keywords: Methanobacterium thermoautotrophicum; structural genomics; heteronuclear NMR; + new fold; MinE protein; cell division
Abbreviations: COG, cluster of ortholog groups • NOG, nonsupervised orthologous group • HSQC, heteronuclear single quantum correlation • HNHB, 3D NMR experiment directed to detect scalar cross-correlation between the amide and H' protons of a given residue.
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04620504.
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Introduction |
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TOPAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
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-strands, which could be arranged in a fold not previously described. Mth677 can not be related to any previously characterized protein, and its function is unknown. However, a BLAST search indicates that it is conserved, with high sequence homology to three hypothetical proteins present in the genomes of three other archaeal thermophiles (Fig. 1
). These proteins are Ph0533 from Pyrococcus horikossii, Pab1365 from Pyrococcus abyssi, and Pf0383 from Pyrococcus furiosus. The sequences of the four proteins are about 25% identical and 50% similar, and the prediction of their secondary structure elements is essentially the same as that for Mth677. The four proteins have been classified as NOG06340 a nonsupervised or-thologous group of proteins (von Mering et al. 2003). To contribute to the effort in determining the structures of the non-membrane associated proteins of Mth and to look for clues that may unveil the functional role of this conserved protein from its structure, we have determined its three-dimensional structure in solution by NMR spectroscopy.
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Results |
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) and side chain assignments (1H, 15N, and 13C) have been obtained for all Mth677 residues with the following exceptions: the nitrogen of Pro68, the 13C carbonyl of the residue preceding Pro68, the 13C
and 13C
of Phe89, the amine protons of lysines, and the protons of guanidinium groups of arginines. The assignment for the additional tag residues Gly-Ser-His is only partial. Nine pairs of methylene H resonances were stereospecifically assigned using HNHB and nuclear overhauser effect (NOE) information. A summary of the consensus chemical shift index, sequential and short-range NOEs, and amide proton exchange raw data is given in Figure 2. According to the whole set of NMR parameters, the elements of secondary structure are well delineated: two helices spanning residues 8–29 and 69–90 and two -strands covering residues 36–46 and 49–60, in satisfactory agreement with the results of most predictive approaches. The assignments have been deposited in the BioMagResBank under accession number 5704
[BMRB]
.
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chemical shifts with the program TALOS (Cornilescu et al. 1999), in concert with 3JHN-H coupling constants. 16 
1 angle constraints were obtained for those residues with stereospecific methylene H assignment. Hydrogen exchange rates, together with preliminary structures, allowed the identification of 22 hydrogen bonds belonging to secondary structure elements. These bonds were included as 44 distance restraints (plus the corresponding 44 lower-bound distance restraints). These contraints were used to generate three-dimensional model structures of the protein, excluding the three tag residues at the N terminus. Preliminary structures were used for automatic assignment of 54 additional interresidue proton distance restraints with CANDID (Herrmann et al. 2002), which were included in the last round of structure calculation and refinement. The only peptide bond preceding a proline residue (Pro 69) was found to be in the trans conformation based on the chemical shifts of the proline carbons (Schubert et al. 2002) as well as on the sequential NOEs. Table 1 summarizes the structural statistics of the final ensemble of 30 refined structures. The final ensemble of 30 refined structures has been deposited in the Protein Data Bank under accession code 1pu1.
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(Figs. 2
, 3). The two helices span residues 8–29 and 69–90 and are packed in an antiparallel way, making an angle of 140°. Two -strands form a long hairpin from V36 to L60 that packs against the two helices. A single residue (D48), with average backbone dihedral angles typical of a 
-turn, and the only nonglycine residue with dihedral angle 
> 0 in all the calculated conformers, connects the two antiparallel -strands. This residue, together with the first two amino acids at the N terminus and the last two at the C terminus have heteronuclear 15N {1H} NOEs smaller than 0.6 (data not shown), indicating high mobility at these sites. Other regions of the chain that connect the secondary structure elements adopt nonregular but well-defined structures, although with larger mobility than the secondary structure elements.
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) and conserved in the four orthologs. The sequence of Mth677 has a large proportion of charged residues, most of them conserved, with their side chains located on the exterior of the protein surface or at the chain termini. These side chains are, in general, not well defined in the family of 30 NMR conformers, but inspection of the structure indicates that at least three ion pairs could be formed, one in each helix and the other one in the hairpin. Although the intrinsic thermal stability of a protein cannot be explained by a single factor, the tightly packed hydrophobic core and these ion pairs would certainly contribute to the thermal stability of Mth677. The melting of the structure monitored by circular dichroism at 222 nm is still not complete at 95°C (data not shown). From that curve, a melting temperature of about 80°C can be estimated.
The acidic amino acids (24% of the total) largely outnumber the basic ones (8%), yielding a theoretical pI of 4.0. The charge is asymmetrically distributed on the protein surface, with a greater density of negative charge on the exposed surface of the hairpin than on the helices’ surface (Fig. 3 and Fig. 6, below). It is difficult to extract a conclusion from this finding, but it is tempting to think that it must be related to the protein function, although it is not obvious what the function can be for a protein with this charge distribution.
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). A similar search with DALI (Holm and Sander 1993; Dietmann et al. 2001) yielded five PDB structures with a z score >5. In all them, the fold of Mth677 was matched to a supersecondary structural element of a larger domain with more than two strands in the -sheet. However, the structure of MinE protein is not selected by DALI as similar to Mth677, probably because of the different approaches used by both methods (contact map alignment in DALI and C trace superposition in MAMMOTH). |
Discussion |
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and proteins (+ ) class, defined by the presence of mainly antiparallel -sheets and segregated and regions in the Structural Classification of Proteins (Murzin et al. 1995). This class contains several folds and superfamilies, among them the fold and superfamily named head-to-tail joining protein W, represented by a single protein, the protein W of bacteriophage 
(Maxwell et al. 2002). This protein contains two antiparallel -helices and a -hairpin, like Mth677, but the two helices are packed against the opposite side of the hairpin (see Fig. 4). The two folds are different: They are mirror images of the arrangement of the elements of secondary structure (A. Murzin, pers. comm.). Although there is a significant structure similarity between MinE and Mth677, as evaluated by MAMMOTH, MinE is a homodimer whose global structure arises from the packing of secondary structure elements from each monomer (Fig. 5). The structure of Mth677 represents, then, a novel fold that is likely to be shared by its three identified orthologs.
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Besides its three orthologs of unknown function, Mth677 is homologous to segments of proteins identified in the fruit fly, mouse, and human genomes, but with no functional annotation. The Web server STRING (von Mering et al. 2003), which uses conservation of neighborhood across genomes as the main criterion to predict functional associations, suggests as possible functions t-RNA binding and prefoldin or chaperonin cofactor. Considering the low pI of Mth677 and the distribution of charges on its surface, it is unlikely that this protein directly binds nucleic acids, although it could regulate the binding of other proteins. On the other hand, the archaeal prefoldin is a hetero hexameric protein that acts as a molecular chaperone stabilizing proteins and releasing them for subsequent chaperonin-assisted folding (Leroux et al. 1999), very different from the small monomeric Mth677. The three-dimensional structure of the protein allows us to look for local structural patterns associated with a particular function. The PINTS server (Stark and Russell 2003) was used to explore the presence of these patterns. Using different NMR models, some hits were found, but they were very diverse and had very high E values typical of negative matches, according to the authors.
Possible role of Mth677 in cell division
By applying the fold recognition program MAMMOTH (Ortiz et al. 2002), a match of part of the Mth677 structure (the N-terminal helix and the -hairpin) with one subunit of the homodimer formed by the C-terminal domain of MinE was found. The MinE protein is involved in cell division and, more specifically, in securing that division takes place at the preferred midcell site. As a previous step to cell division, the tubulin-like protein FtsZ forms a circumferential ring (septum) at the division site. The complex formed by two other proteins, MinC and MinD, inhibits cell division by blocking the ring septum assembly. MinE forms an annular structure near the middle of the cell, suppressing the activity of the complex MinCD and allowing FtsZ ring formation at midcell (Margolin 2001). The 88-residue MinE protein of E. coli contains two separable functional domains. The 32-residue N-terminal counteracts MinCD activity, and the C-terminal domain (MinETSD) controls the topological specificity for midcell localization of MinE. MinETSD forms in solution a homodimer, whose global structure consists of an antiparallel coiled coil, packed against one face of a four-stranded sheet (King et al. 2000).
As mentioned above, the N-terminal helix and the -hairpin of Mth677 match very closely with one subunit of MinETSD, and even the C-terminal helix of Mth677 occupies the same position and orientation as the helix in the second subunit of the homodimer of MinETSD (Fig. 5). Both proteins appear as more or less cylindrical when viewed from their longest axis (see Fig. 3, bottom, for Mth677; and Fig. 2 in King et al. 2000 for MinETSD). It is also worth noting the large negative electrostatic potential of both proteins and the similarity of its distribution, in particular the large negative potential on the solvent-exposed surface of the -sheet (see Fig. 6). It is reasonable to speculate that both shape and charge might be required to form the annular structure by side-by-side juxtaposition of a number of these modular proteins and/or by interaction with the internal side of the membrane. In that case, the annular structure formed by Mth677 and the homodimeric MinETSD would be very similar, and then Mth677 would be the equivalent of MinETSD in Mth. A fact in favor of that proposal is that there is no homolog of MinETSD in the genome of Mth, and that homologs of MinD and FtsZ are, however, found. On the other hand, no clear homologs of MinC and of the N-terminal domain of MinE were found. The proposal that Mth677 might be, in Mth, the counterpart of MinETSP in E. coli needs to be further confirmed. A right step in that direction would be to observe the consequences of deleting the implied gene on cell division.
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Materials and methods |
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DE3) cells cotransformed with a plasmid encoding three transfer RNAs for rare E. coli codons and grown at 37°C until the OD600 nm reached 0.6. After protein expression induction with 1 mM isopropyl -D-thiogalactoside, the temperature was reduced to 15°C and the cells were allowed to grow overnight before harvesting. Frozen cell pellets were thawed in 500 mM NaCl/20 mM Tris/5 mM imidazole (pH 8) and lysed by sonication. The protein was extracted by batch Ni2+ affinity chromatography (Qiagen), and the affinity beads were washed three times with five column volumes of 500 mM NaCl/20 mM Tris/30 mM imidazole (pH 8); the protein was eluted with five column volumes of the same buffer plus 500 mM imidazole. The His tag was removed by cleavage with thrombin and the protein purified by anion exchange chromatography using DEAE Sepharose. The purified protein was concentrated, and the buffer was exchanged by ultrafiltration and dilution/reconcentration. The final 94 residue long polypeptide analyzed here contains the sequence of Mth677 protein plus three extraneous residues (GSH) at its N-terminus, coming from the thrombin cleavage site. Equilibrium sedimentation analysis indicates that Mth677 is a monomer under the conditions used for the acquisition of the NMR spectra (data not shown). Its circular dichroism spectrum is typical of a protein with mixed and secondary structure. Three labeled samples were prepared growing bacteria in M9 minimal medium supplemented with 15NH4Cl or with 15NH4Cl and [13C6] glucose U-15N, U-13C,15N, and U-13C,15N fully exchanged in 2H2O.
NMR spectra
NMR experiments were recorded on a Bruker AVANCE 600 spectrometer with a triple resonance (1H, 15N, and 13C) z-gradient probe at 298 K. NMR samples were prepared with a protein concentration of approximately 1.2 mM in 25 mM sodium phosphate buffer (pH 7.0), 0.45 M NaCl, 10 mM DTT, 20 µM ZnCl2, 1 mM benzamidine, and a protease inhibitor mixture (Roche Molecular Biochemicals). Some spectra were also recorded at 278 K to assign overlapping resonances.
For the sequential assignment of backbone resonances HNCACB, CBCA(CO)NH, HNHA, and HNCO experiments (Bax and Grzesiek 1993) were acquired and analyzed with reference to the 1H-15N HSQC spectrum. Figure 7 shows the 1H-15N HSQC spectrum as a sample of the spectral quality. The following experiments were performed for the assignment of nuclear resonances in side chains: HNHB (Bax et al. 1994); 2D 1H-1H NOESY and TOCSY with mixing times of 120 and 80 msec, respectively; 3D 15N edited NOESY and TOCSY (same mixing times as the 2D) (Bax et al. 1990; Grzesiek et al. 1993); two different CT-1H-13C HSQC, optimized for correlations involving either aliphatic or aromatic protons; 3D (H)C(CCO)NH (15.2 msec carbon mixing time using the DIPSI sequence); and HCCH-TOCSY (Bax et al. 1994; 18 msec mixing time). The aromatic side chains were assigned to specific residues from their NOEs with the CH2 groups and the backbone amide protons.
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Structure determination
The NMR constraints were used as input for the torsion angle dynamics program CYANA (Güntert et al. 1997) to generate three-dimensional model structures of the protein, excluding the three tag residues at the N terminus. The structures were refined by energy minimization with the AMBER 7.0 package (Case et al. 2002). In this step, all distance restraints involving methyl and nonstereospecifically assigned protons were used with an average of r–6, and additional constraints to keep the planarity of the backbone peptide bonds and the chirality of the amino acids were included.
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Acknowledgments |
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The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
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References |
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Bax, A., Clore, G.M., and Gronenborn, A.M. 1990. 1H-1H Correlation via isotropic mixing of 13C magnetization, a new three-dimensional approach for assigning 1H and 13C spectra of 13C enriched proteins. J. Mag. Reson. 88: 425–431.
Bax, A., Vuister, G.W., Grzesiek, S., Delaglio, F., Wang, A.C., Tschudin, R., and Zhu, G. 1994. Measurement of homo- and heteronuclear J couplings from quantitative J correlation. Methods Enzymol. 239: 79–105.[Medline]
Brenner, S.E., Koehl, P., and Levitt, M. 2000. The ASTRAL compendium for protein structure and sequence analysis. Nucleic Acids Res. 28: 254–256.
Case, D.A., Pearlman, D.A., Caldwell, J.W., Cheatham III, T.E., Wang, J., Ross, W.S., Simmerling, C.L., Darden, T.A., Merz, K.M., et al. 2002. AMBER 7.0. University of California, San Francisco.
Chandonia, J.M., Walker, N.S., Lo Conte, L., Koehl, P., Levitt, M., and Bren-ner, S.E. 2002. ASTRAL compendium enhancements. Nucleic Acids Res. 30: 260–263.
Christendat, D., Yee, A., Dharamsi, A., Kluger, Y., Savchenko, A., Cort, J.R., Booth, V., Mackereth, C.D., Saridakis, V., Ekiel, I., et al. 2000. Structural proteomics of an archaeon. Nat. Struct. Biol. 7: 903–909.[CrossRef][Medline]
Cornilescu, G., Delaglio, F., and Bax, A. 1999. Protein backbone angle restraints from searching a database for chemical shift and sequence homology. J. Biomol. NMR 13: 289–302.[CrossRef][Medline]
Delaglio, F., Grzesiek, S., Vuister, G.W., Zhu, G., Pfeifer, J., and Bax, A. 1995. NMRPipe: A multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6: 277–293[Medline]
Dietmann, S., Park, J., Notredame, C., Heger, A., Lappe, M., and Holm, L. 2001. A fully automatic evolutionary classification of protein folds: Dali Domain Dictionary version 3. Nucleic Acids Res. 29: 55–57
Grzesiek, S., Anglister, A., and Bax, A. 1993. Correlation of backbone and aliphatic side-chain resonances in 13C/15N-enriched proteins by isotropic mixing of 13C magnetization. J. Mag. Reson. 101: 114–119.
Güntert, P., Mumenthaler, C., and Wüthrich, K. 1997. Torsion angle dynamics for NMR structure calculation with the new program DYANA. J. Mol. Biol. 273: 283–298.[CrossRef][Medline]
Herrmann, T., Güntert, P., and Wüthrich, K. 2002. Protein NMR structure determination with automated NOE assignment using the new software CANDID and the torsion angle dynamics algorithm DYANA. J. Mol. Biol. 319: 209–227.[CrossRef][Medline]
Holm, L. and Sander, C. 1993. Protein structure comparison by alignment of distance matrices. J. Mol. Biol. 233: 123–138[CrossRef][Medline]
Johnson, B. and Blevins, R.A. 1994. NMRView: A computer program for the visualization and analysis of NMR data. J. Biomol. NMR 4: 603–614.[CrossRef]
King, G.F., Shih, Y.L., Maciejewski, M.W., Bains, N.P., Pan, B., Rowland, S.L., Mullen, G.P., and Rothfield, L.I. 2000. Structural basis for the topological specificity function of MinE. Nat. Struct. Biol. 7: 1013–1017.[CrossRef][Medline]
Koradi, R., Billeter, M., and Wuthrich, K. 1996. MOLMOL: A program for display and analysis of macromolecular structures. J. Mol. Graph. 14: 51–55.[CrossRef][Medline]
Laskowski, R.A., Rullmannn, J.A., MacArthur, M.W., Kaptein, R., and Thornton, J.M. 1996. AQUA and PROCHECK-NMR: Programs for checking the quality of protein structures solved by NMR. J. Biomol. NMR 8: 477–486.[Medline]
Leroux, M.R., Fandrich, M., Klunker, D., Siegers, K., Lupas, A.N., Brown, J.R., Schiebel, E., Dobson, C.M., and Hartl, F.U. 1999. MtGimC, a novel archaeal chaperone related to the eukaryotic chaperonin cofactor GimC/prefoldin. EMBO J. 18: 6730–6743.[CrossRef][Medline]
Lo Conte, L., Brenner, S.E., Hubbard, T.J., Chothia, C., and Murzin, A.G. 2002. SCOP database in 2002: Refinements accommodate structural genomics. Nucleic Acids Res. 30: 264–267.
Margolin, W. 2001. Bacterial cell division: A moving MinE sweeper boggles the MinD. Curr. Biol. 11: R395–398.[CrossRef][Medline]
Maxwell, K.L., Yee, A.A., Arrowsmith, C.H., Gold, M., and Davidson, A.R. 2002. The solution structure of the bacteriophage head-tail joining protein, gpFII. J. Mol. Biol. 318: 1395–1404.[CrossRef][Medline]
Murzin, A.G., Brenner, S.E., Hubbard, T., and Chothia, C. 1995. SCOP: A structural classification of proteins database for the investigation of sequences and structures. J. Mol. Biol. 247: 536–540.[CrossRef][Medline]
Ortiz, A.R., Strauss, C.E., and Olmea, O. 2002. MAMMOTH (matching molecular models obtained from theory): An automated method for model comparison. Protein Sci. 11: 2606–2621.
Shubert, M., Labudde, D., Oschkinat, H., and Schmieder, P. 2002. A software tool for the prediction of Xaa-Pro peptide bond conformations in proteins based on 13C chemical shifts. J. Biomol. NMR 24: 149–154.[CrossRef][Medline]
Stark, A. and Russell, R.B. 2003. Annotation in three dimensions. PINTS: patterns in non-homologous tertiary structures. Nucleic Acids Res. 31: 3341–3344.
Wishart, D.S. and Sykes, B.D. 1994. The 13C chemical-shift index: A simple method for the identification of protein secondary structure using 13C chemical-shift data. J. Biomol. NMR 4: 171–180.[Medline]
Wishart, D.S., Bigam, C.G., Yao, J., Abildgaard, F., Dyson, H.J., Oldfield, E., Markley, J.L., and Sykes, B.D. 1995. 1H, 13C, and 15N chemical shift referencing in biomolecular NMR. J. Biomol. NMR 6: 135–140.[Medline]
von Mering, C., Huynen, M., Jaeggi, D., Schmidt, S., Bork, P., and Snel, B. 2003. STRING: A database of predicted functional associations between proteins. Nucleic Acids Res. 31: 258–261.
Yee, A., Chang, X., Pineda-Lucena, A., Wu, B., Semesi, A., Le, B., Ramelot, T., Lee, G. M., Bhattacharyya, S., Gutierrez, P., et al. 2002. An NMR approach to structural proteomics. Proc. Natl. Acad. Sci. 99: 1825–1830.
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