Protein Science Sheba protein
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Published online before print January 10, 2004, 10.1110/ps.03401004
Protein Science (2004), 13:545-548. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
ps.03401004v1
13/2/545    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kasai, T.
Right arrow Articles by Yokoyama, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kasai, T.
Right arrow Articles by Yokoyama, S.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

FOR THE RECORD

Solution structure of a BolA-like protein from Mus musculus

Takuma Kasai1,2, Makoto Inoue1, Seizo Koshiba1, Takashi Yabuki1, Masaaki Aoki1, Emi Nunokawa1, Eiko Seki1, Takayoshi Matsuda1, Natsuko Matsuda1, Yasuko Tomo1, Mikako Shirouzu1,3, Takaho Terada1,3, Naomi Obayashi1, Hiroaki Hamana1, Naoko Shinya1, Ayako Tatsuguchi1, Satoko Yasuda1, Mayumi Yoshida1, Hiroshi Hirota1, Yo Matsuo1, Kazutoshi Tani1, Harukazu Suzuki1, Takahiro Arakawa1, Piero Carninci1, Jun Kawai1, Yoshihide Hayashizaki1, Takanori Kigawa1 and Shigeyuki Yokoyama1,2,3

1 RIKEN Genomic Sciences Center, Yokohama, Japan
2 Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Tokyo, Japan
3 RIKEN Harima Institute at SPring-8, Hyogo, Japan

Reprint requests to: Shigeyuki Yokoyama, Protein Research Group, RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan; e-mail: yokoyama{at}biochem.s.u-tokyo.ac.jp; fax: 81-45-503-9195.


   Abstract
TOP
 Abstract
Introduction
 Results and Discussion
 Materials and methods
 References
 
The BolA-like proteins are widely conserved from prokaryotes to eukaryotes. The BolA-like proteins seem to be involved in cell proliferation or cell-cycle regulation, but the molecular function is still unknown. Here we determined the structure of a mouse BolA-like protein. The overall topology is {alpha}{beta}{beta}{alpha}{alpha}{beta}{alpha}, in which {beta}1 and {beta}2 are antiparallel, and {beta}3 is parallel to {beta}2. This fold is similar to the class II KH fold, except for the absence of the GXXG loop, which is well conserved in the KH fold. The conserved residues in the BolA-like proteins are assembled on the one side of the protein.

Keywords: NMR structure; conserved protein; BolA; cell proliferation; cell-cycle regulation; KH fold; structural genomics

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03401004.


   Introduction
TOP
 Abstract
 Introduction
Results and Discussion
 Materials and methods
 References
 
Escherichia coli BolA and its homologs constitute a widely conserved protein family, called the BolA-like protein family. BolA-like proteins are found in most bacteria and eukaryotes, and in an archaeon. The E. coli bolA gene was isolated as the morphogene induced in stationary phase (Aldea et al. 1988, 1989, 1990; Lange and Hengge-Aronis 1991), and was also found to be induced by various stresses (Santos et al. 1999). The mutation or the overexpression of the bolA gene affects the transcription level of the penicillin binding protein (PBP) genes, which are involved in cell wall biosynthesis (Aldea et al. 1989; Santos et al. 2002). In Schizosaccharomyces pombe, a bolA homolog, uvi31+, which was isolated as a UV-inducible gene, shows cell-cycle and growth phase-dependent expression (Lee et al. 1994; Kim et al. 1997). In addition, the deletion mutant of uvi31 proliferates faster than the wild-type cell, and shows abnormal septation after UV-induced cell cycle arrest (Kim et al. 2002). Although these results imply that BolA-like proteins play some roles in cell proliferation by controlling the transcription of other genes, their molecular functions remain unknown.

Most of the BolA-like proteins consist of about 100 amino acid residues, as do the three BolA-like proteins found within the mouse full-length cDNA libraries, FANTOM and FANTOM2 (Kawai et al. 2001; Okazaki et al. 2002). We designated the mouse BolA-like proteins as BolA1 (gi|12841442), BolA2 (gi|26325949), and BolA3 (gi|26389531). Among the three proteins, BolA1 shows the highest homology to E. coli BolA. Close homologs of the mouse BolA2 were found in most eukaryotes, including Homo sapiens, Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana, and Saccharomyces cerevisiae. Therefore, the BolA2 proteins constitute a eukaryotic subfamily of the BolA-like proteins (Fig. 1AGo). Mouse BolA3 and its homologs compose another eukaryotic subfamily, with lower similarity to E. coli BolA than the BolA2 subfamily.



View larger version (53K):
[in this window]
[in a new window]
  		Figure 1. (A) Multiple sequence alignment of the BolA2 subfamily. Highlighted and shaded residues indicate identical and similar residues, respectively. Mm: BolA2 from Mus musculus. Hs: My016 from Homo sapiens. Dm: CG16804-PB from Drosophila melanogaster. Ag: agCP4532 from Anopheles gambiae. Sp: SPAC8C9.11 from Schizosaccharomyces pombe. The multiple alignment was achieved with CLUSTAL_X (Thompson et al. 1997). The secondary structures of the mouse BolA2 are indicated at the top of the alignment. (B) Ribbon diagram of BolA2. (C) Class II KH domains of ribosomal protein S3 (top, PDB ID: 1J5E [PDB] ) and Era GTPase (bottom, PDB ID: 1EGA [PDB] ). Each class II KH domain is colored in the rainbow order, from red (N terminus) to blue (C terminus).

 
In the present study, we determined the solution structure of the mouse BolA2, which is the first tertiary structure of a BolA-like protein family member. The BolA2 protein has the {alpha}{beta}{beta}{alpha}{alpha}{beta}{alpha} topology, which is the same as that of the class II KH fold. However, the BolA-like proteins lack the GXXG sequence, which is involved in binding to nucleic acids in the authentic KH-fold proteins. Instead of the GXXG loop, BolA2 has a turn, which results in a helix-turn-helix (HTH) motif. The surface residues around the HTH motif are well conserved in the BolA2 subfamily.


   Results and Discussion
TOP
 Abstract
 Introduction
 Results and Discussion
Materials and methods
 References
 
The overall topology of BolA2 is {alpha}{beta}{beta}{alpha}{alpha}{beta}{alpha}, in which {beta}1 and {beta}2 are antiparallel, and {beta}3 is parallel to {beta}2 (Fig. 1BGo). A nine-residue loop, inserted between {beta}1 and {beta}2, has several amide protons with very low intensities in the 1H/15N-HSQC spectrum. The {alpha}4 helix is anchored on one side of the {beta}-sheet, while the other helices are on the other side. A one-turn 310-like helix exists between {alpha}3 and {beta}3. The {alpha}2 and {alpha}3 helices form an HTH motif, and are arranged at an angle of about 60° to each other. In E. coli BolA, another HTH motif had been predicted by the Chou-Fasman algorithm (Aldea et al. 1989). However, the corresponding region of BolA2 comprises the {beta}1/{beta}2 loop and the {beta}2 strand.

According to the DALI (Holm and Sander 1993) search, BolA2 shows significant structural similarity to the ribosomal protein S3 N-terminal domain (Wimberly et al. 2000) and the Era GTPase C-terminal domain (Chen et al. 1999), with Z-values of 5.4 and 4.7, respectively, which belong to class II KH fold (Fig. 1CGo). The KH fold is commonly found among nucleic acid binding proteins, and consists of two classes with distinct topologies, {beta}{alpha}{alpha}{beta}{beta}{alpha} for class I and {alpha}{beta}{beta}{alpha}{alpha}{beta} for class II (Grishin 2001). Most of the KH-fold proteins have a well-conserved GXXG sequence on the loop between two adjacent helices. Some of the class I KH-fold proteins bind to nucleic acids using their GXXG loop (Lewis et al. 2000; Liu et al. 2001; Braddock et al. 2002a,b). The corresponding region of BolA2 forms an HTH motif, and the BolA-like proteins lack the conserved GXXG sequence in this region.

Figure 2AGo shows the conserved surface residues of BolA2. Most of the conserved residues are assembled on one side of the protein, especially around the HTH motif. The "conserved" side consists of an electrically neutral region surrounded by several basic residues (Fig. 2BGo). There are significantly conserved solvent-exposed hydrophobic residues (L50, L55) near the HTH motif. On the other hand, the other side of BolA2 is highly acidic. Some residues are still conserved on the "variable" side of BolA2.



View larger version (48K):
[in this window]
[in a new window]
  		Figure 2. (A) The conserved residues on the surface of the mouse BolA2. The identical and the similar residues defined in Figure 1AGo are colored dark blue and light blue, respectively. The right panel is viewed from the opposite side of the left panel. (B) The surface electrostatic potential of BolA2. (C) Ribbon diagram in the same orientation as A and B. The HTH motif is indicated by the blue circle.

 
The nucleic-acid binding property of the BolA-like proteins is implicated by the structural similarity to the KH fold and the assembly of conserved surface residues around the HTH motif. However, the existence of some conserved surface residues on the "variable" side and the absence of the GXXG sequence suggest the possibility that the BolA-like proteins have other functions than those of the KH-fold proteins.


   Materials and methods
TOP
 Abstract
 Introduction
 Results and Discussion
 Materials and methods
References
 
Sample preparation and NMR spectroscopy
The mouse BolA2 protein was produced as the 113-amino-acid recombinant protein with a 27-amino-acid purification tag sequence (MKGSSHHHHH HSSGASLVPR GSEGAAT) at the N terminus of the 86-amino-acid coding sequence. The uniformly 13C- and 15N-labeled protein was produced by the E. coli cell-free protein synthesis system (Kigawa et al. 1999), and then 5.0 mg of the purified protein were obtained by polyhistidine affinity chromatography followed by anion exchange chromatography. All NMR spectra were acquired on a Bruker AVANCE600 spectrometer at 298K, with a 1.2 mM 13C/15N BolA2 protein solution in 50 mM Na-phosphate buffer (pH 6.0) containing 100 mM NaCl, 0.02% NaN3, 1 mM DTT-d10, and 10% 2H2O. Spectra were processed and analyzed with NMRPipe (Delaglio et al. 1995) and NMRview (Johnson and Blevins 1994).

Structure determinaiton
The chemical shifts were assigned using 1H/15N-HSQC, 1H/13C-HSQC, HNCA, HN(CO)CA, HNCACB, CBCA(CO)NH, HNCO, HN(CA)CO, HBHA(CO)NH, CC(CO)NH, H(CCCO)NH, HCCH-COSY, HCCH-TOCSY, NOESY-1H/15N-HSQC ({tau}m = 100 msec), and NOESY-1H/13C-HSQC ({tau}m = 100 msec) spectra. In the BolA2 coding region, 97% of the observable proton signals were assigned. The NOESY cross-peaks were assigned with DYANA/CANDID (Herrmann et al. 2002), following manual correction. The total number of assigned NOEs was 1546, but 931 meaningful distance constraints were used for the final structure calculation. The dihedral-angle constraints of the main chain were used in the secondary structure regions predicted by TALOS (Cornilescu et al. 1999; {phi} = -60° ± 15°, {psi} = -45° ± 15° for the helical regions, {phi} = -120° ± 30°, {psi} = 150° ± 30° for the {beta}-strand regions). The side-chain {chi} angle constraints (180°, 60°, or -60° with a tolerance of ±30°) were used for the residues for which the {chi} angles could be obviously determined from the NOESY spectra. To obtain hydrogen bond information, 1H/15N-HSQC spectra were measured in 2H2O. Twenty-nine peaks in the {alpha}-helix or the {beta}-strand regions were used for the structure calculation, while a total of 37 peaks were observed. Hydrogen bond constraints were used as two distance constraints (3.5 Å for N and O, 2.5 Å for HN and O) for each hydrogen bond. The final structure was calculated with CNS (Brünger et al. 1998). The calculation statistics are summarized in Table 1Go. The coordinates of the best 20 structures have been deposited in the Protein Data Bank, under the accession ID 1IW5 [PDB] . The ribbon diagrams were drawn with MOLMOL (Koradi et al. 1996). The molecular surface presentations were generated by GRASP (Nicholls et al. 1991).


View this table:
[in this window]
[in a new window]
  		Table 1. Calculation statistics for the ensemble of 20 structures
 


   Acknowledgments
 
We are grateful to Yukiko Fujikura, Yoko Motoda, Miyuki Saito, Yukako Miyata, Atsuo Kobayashi, Noriko Hirakawa, Noriko Sakagami, Masaomi Ikari, Fumiko Hiroyasu, and Megumi Watanabe for their technical assistance. This work was supported by the RIKEN Structural Genomics/Proteomics Initiative (RSGI), the National Project on Protein Structural and Functional Analyses, Ministry of Education, Culture, Sports, Science and Technology of Japan.

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.


   References
TOP
 Abstract
 Introduction
 Results and Discussion
 Materials and methods
 References
 
Aldea, M., Hernández-Chico, C., de la Campa, A.G., Kushner, S.R., and Vicente, M. 1988. Identification, cloning, and expression of bolA, an ftsZ-dependent morphogene of Escherichia coli. J. Bacteriol. 170: 5169–5176.[Abstract/Free Full Text]

Aldea, M., Garrido, T., Hernández-Chico, C., Vicente, M., and Kushner, S.R. 1989. Induction of a growth-phase-dependent promoter triggers transcription of bolA, an Escherichia coli morphogene. EMBO J. 8: 3923–3931.[Medline]

Aldea, M., Garrido, T., Pla, J., and Vicente, M. 1990. Division genes in Escherichia coli are expressed coordinately to cell septum requirements by gearbox promoters. EMBO J. 9: 3787–3794.[Medline]

Braddock, D.T., Louis, J.M., Baber, J.L., Levens, D., and Clore, G.M. 2002a. Structure and dynamics of KH domains from FBP bound to single-stranded DNA. Nature 415: 1051–1056.[CrossRef][Medline]

Braddock, D.T., Baber, J.L., Levens, D., and Clore G.M. 2002b. Molecular basis of sequence-specific single-stranded DNA recognition by KH domains: Solution structure of a complex between hnRNP K KH3 and single-stranded DNA. EMBO J. 21: 3476–3485.[CrossRef][Medline]

Brünger, A.T., Adams, P.D., Clore, G.M., DeLano, W.L., Gros, P., Grosse-Kunstleve, R.W., Jiang, J.S., Kuszewski, J., Nilges, M., Pannu, N.S., et al. 1998. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54: 905–921.[CrossRef][Medline]

Chen, X., Court, D.L., and Ji, X. 1999. Crystal structure of ERA: A GTPase-dependent cell cycle regulator containing an RNA binding motif. Proc. Natl. Acad. Sci. 96: 8396–8401.[Abstract/Free Full Text]

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]

Grishin, N.V. 2001. KH domain: One motif, two folds. Nucleic Acids Res. 29: 638–643.[Abstract/Free Full Text]

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.A. and Blevins, R.A. 1994. NMRView: A computer program for the visualization and analysis of NMR data. J. Biomol. NMR 4: 603–614.[CrossRef]

Kawai, J., Shinagawa, A., Shibata, K., Yoshino, M., Itoh, M., Ishii, Y., Arakawa, T., Hara, A., Fukunishi, Y., Konno, H., et al. 2001. Functional annotation of a full-length mouse cDNA collection. Nature 409: 685–690.[CrossRef][Medline]

Kigawa, T., Yabuki, T., Yoshida, Y., Tsutsui, M., Ito, Y., Shibata, T., and Yokoyama, S. 1999. Cell-free production and stable-isotope labeling of milligram quantities of proteins. FEBS Lett. 442: 15–19.[CrossRef][Medline]

Kim, S.H., Kim, M., Lee, J.K., Kim, M.J., Jin, Y.H., Seong, R.H., Hong, S.H., Joe, C.O., and Park, S.D. 1997. Identification and expression of uvi31+, a UV-inducible gene from Schizosaccharomyces pombe. Environ. Mol. Mutagen. 30: 72–81.[CrossRef][Medline]

Kim, M.J., Kim, H.S., Lee, J.K., Lee, C.B., and Park, S.D. 2002. Regulation of septation and cytokinesis during resumption of cell division requires uvi31+, a UV-inducible gene of fission yeast. Mol. Cells 14: 425–430.[Medline]

Koradi, R., Billeter, M., and Wüthrich, K. 1996. MOLMOL: A program for display and analysis of macromolecular structures. J. Mol. Graphics 14: 51–55.[CrossRef][Medline]

Lange, R. and Hengge-Aronis, R. 1991. Growth phase-regulated expression of bolA and morphology of stationary-phase Escherichia coli cells are controlled by the novel sigma factor {sigma}S. J. Bacteriol. 173: 4474–4481.[Abstract/Free Full Text]

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]

Lee, J.K., Park, E.J., Chung, H.K., Hong, S.H., Joe, C.O., and Park, S.D. 1994. Isolation of UV-inducible transcripts from Schizosaccharomyces pombe. Biochem. Biophys. Res. Commun. 202: 1113–1119.[CrossRef][Medline]

Lewis, H.A., Musunuru, K., Jensen, K.B., Edo, C., Chen, H., Darnell, R.B., and Burley, S.K. 2000. Sequence-specific RNA binding by a Nova KH domain: Implications for paraneoplastic disease and the fragile X syndrome. Cell 100: 323–332.[CrossRef][Medline]

Liu, Z., Luyten, I., Bottomley, M.J., Messias, A.C., Houngninou-Molango, S., Sprangers, R., Zanier, K., Krämer, A., and Sattler, M. 2001. Structural basis for recognition of the intron branch site RNA by splicing factor 1. Science 294: 1098–1102.[Abstract/Free Full Text]

Nicholls, A., Sharp, K.A., and Honig, B. 1991. Protein folding and association: Insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins 11: 281–296.[CrossRef][Medline]

Okazaki, Y., Furuno, M., Kasukawa, T., Adachi, J., Bono, H., Kondo, S., Nikaido, I., Osato, N., Saito, R., Suzuki, H., et al. 2002. Analysis of the mouse transcriptome based on functional annotation of 60,770 full-length cDNAs. Nature 420: 563–573.[CrossRef][Medline]

Santos, J.M., Freire, P., Vicente, M., and Arraiano, C.M. 1999. The stationary-phase morphogene bolA from Escherichia coli is induced by stress during early stages of growth. Mol. Microbiol. 32: 789–798.[CrossRef][Medline]

Santos, J.M., Lobo, M., Matos, A.P., De Pedro, M.A., and Arraiano, C.M. 2002. The gene bolA regulates dacA (PBP5), dacC (PBP6) and ampC (AmpC), promoting normal morphology in Escherichia coli. Mol. Microbiol. 45: 1729–1740.[CrossRef][Medline]

Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., and Higgins, D.G. 1997. The CLUSTAL_X windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25: 4876–4882.[Abstract/Free Full Text]

Wimberly, B.T., Brodersen, D.E., Clemons Jr., W.M., Morgan-Warren, R.J., Carter, A.P., Vonrhein, C., Hartsch, T., and Ramakrishnan, V. 2000. Structure of the 30S ribosomal subunit. Nature 407: 327–339.[CrossRef][Medline]


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?



This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
ps.03401004v1
13/2/545    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kasai, T.
Right arrow Articles by Yokoyama, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kasai, T.
Right arrow Articles by Yokoyama, S.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS