Structure of Bacillus subtilis YXKO—A member of the UPF0031 family and a putative kinase

doi:10.1016/S1047-8477(02)00532-4

Journal of Structural Biology

Volume 139, Issue 3, September 2002, Pages 161-170

https://doi.org/10.1016/S1047-8477(02)00532-4 Get rights and content

Abstract

We determined the 1.6-Å resolution crystal structure of a conserved hypothetical 29.9-kDa protein from the SIGY–CYDD intergenic region encoded by a Bacillus subtilis open reading frame in the YXKO locus. YXKO homologues are broadly distributed and are by and large described as proteins with unknown function. The YXKO protein has an α/β fold and shows high structural homology to the members of a ribokinase-like superfamily. However, YXKO is the only member of this superfamily known to form tetramers. Putative binding sites for adenosine triphosphate (ATP), a substrate, and Mg²⁺-binding sites were revealed in the structure of the protein, based on high structural similarity to ATP-dependent members of the superfamily. Two adjacent monomers contribute residues to the active site. The crystal structure provides valuable information about the YXKO protein’s tertiary and quaternary structure, the biochemical function of YXKO and its homologues, and the evolution of its ribokinase-like superfamily.

Introduction

The far-reaching goal of structural genomics programs is to map the entire protein folding space. These pilot projects exploit the available genome sequence information to select targets whose structures can be determined. Among genes coding for proteins that show no significant sequence similarity to proteins with known structure, there is a large group of conserved “hypothetical” open reading frames (ORFs)² (Vitkup et al., 2001). Many of these ORFs include members from diverse organisms and can be grouped into large families. Their incidence in many branches of life suggests that they are important; however, their functions cannot be revealed through sequence analysis. Selection of such targets for structural genomics studies is important because the 3D structure of these proteins may offer insight not only into their folds but also into their biochemical or biophysical functions (Zarembinski et al., 1998)—provided that a structural homologue is already known (Martinez-Cruz et al., 2002)—and allow this knowledge to be extended to their sequence homologues. It is anticipated that as the number of nonredundant protein structures grows in the Protein Data Bank (PDB), this approach will be more successful in defining the functions of “hypothetical” proteins.

For this work we have selected, from a large set of structural genomics targets of the gram-positive bacterium Bacillus subtilis (http://www.mcsg.anl.gov), a conserved hypothetical 29.9-kDa protein from the SIGY–CYDD intergenic region encoded by the B. subtilis ORF in the YXKO locus (DNA bases 50795–51625) (Yoshida et al., 1996). [The code “YXKO” will be used for this protein (aka the Midwest Center for Structural Genomics APC234) throughout this text.] Sequence similarity searches of the National Center for Biotechnology Information nonredundant database, using BLAST (Altschul et al., 1997) and FASTA (Pearson, 2000), and the SWISS-PROT (Bairoch and Apweiler, 2000) and Protein Information Resource (Wu et al., 2002) databases resulted in 69 protein homologues of YXKO with statistically significant E values (E<10⁻⁵). These proteins are broadly distributed and are found in 42 species of bacteria, 14 species of archaea, and 13 eukaryotes, including human, and are described as proteins with unknown function. A few are classified as probable sugar kinases (Fig. 1).

These proteins belong to the Pfam (Bateman et al., 1999) UPF0031 family, which includes 62 uncharacterized bacterial proteins and contains two Prosite sequence patterns [UPF0031_1, (SAV)-(IVW)-(LVA)-(LIV)-G-(PNS)-G-L-(GP)-x-(DENQT), and UPF0031_2,(GA)-G-x-G-D-(TV)-(LT)-(STA)-G-x-(LIVM)]. The structures of all these proteins are unknown. YXKO has distant sequence homology with the ThiK protein (PDB entry 1ekk) deposited in the PDB (discussed later in this paper).

We have determined the 1.6-Å resolution crystal structure of YXKO from B. subtilis. The protein shows structural homology to members of the ribokinase-like carbohydrate kinase superfamily, as defined by SCOP (Murzin et al., 1995) and CATH (Orengo et al., 1997). Putative ATP- and substrate-binding sites in the YXKO structure were assigned and they reveal high structural similarity within ATP-dependent ribokinases (RKs). Further elucidation of YXKO’s detailed biochemical function will require a combination of enzymatic, mutational, and structural studies; however, the crystal structure for YXKO provides important insight into the function of this protein and others in the UPF0031 family.

Section snippets

Cloning, expression, and protein purification

The ORF of a B. subtilis YXKO protein (29.9 kDa) in the SIGY–CYDD intergenic region was amplified from genomic DNA with Pfx DNA polymerase using conditions and reagents provided by the vendor (Invitrogen). The gene was cloned into a pMCSG7 vector (Stols et al., 2002) using a modified ligation-independent cloning protocol (Dieckman et al., 2002). This process generated an expression clone producing a fusion protein with an N-terminal His₆ tag and a TEV protease recognition site (ENLYFQ↓S). The

Description of YXKO structure

The protein crystallized in centered tetragonal crystal form in an I422 space group with one subunit in the asymmetric unit. In the crystal the YXKO monomer has an α/β fold and shows structural similarity to the RK-like carbohydrate kinase superfamily (Fig. 2) (Murzin et al., 1995; Orengo et al., 1997). Each subunit of YXKO contains 276 amino acids, with approximately 42% of the amino acids in α helices, 12% in β sheets, and 3% in 3₁₀ helices. The remaining portion of the structure is composed

Acknowledgements

We thank all the members of the Structural Biology Center at Argonne National Laboratory for their help in conducting experiments. This work was supported by National Institutes of Health Grant GM62414 and by the U.S. Department of Energy, Office of Biological and Environmental Research. Atomic coordinates have been deposited with the Protein Data Bank as 1KYH.

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^☆: This article has been created by the University of Chicago as Operator of Argonne National Laboratory under Contract W-31-109-ENG-38 with the US Department of Energy. The US Government’s right to retain a nonexclusive royalty-free license in and to the copyright covering this paper, for governmental purposes, is acknowledged.

¹: Permanent address: Department of Chemistry, Wrocław, University of Technology, Poland.

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Structure of Bacillus subtilis YXKO—A member of the UPF0031 family and a putative kinase☆

Abstract

Introduction

Section snippets

Cloning, expression, and protein purification

Description of YXKO structure

Acknowledgements

Structure (Cambridge)

Trends Biochem. Sci.

Structure (Cambridge)

Structure

J. Mol. Biol.

Methods Enzymol.

Structure

Gapped BLAST and PSI-BLAST: a new generation of protein database search programs

Nucleic. Acids. Res.

The SWISS-PROT protein sequence database and its supplement TrEMBL in 2000

Nucleic. Acids. Res.

Pfam 3.1: 1313 multiple alignments and profile HMMs match the majority of proteins

Nucleic. Acids. Res.

Crystallography & NMR system: a new software suite for macromolecular structure determination

Acta. Crystallogr D Biol. Crystallogr.

Crystal structure of 4-methyl-5-beta-hydroxyethylthiazole kinase from Bacillus subtilis at 1.5 Å resolution

Biochemistry

SUPERFAMILY: HMMs representing all proteins of known structure. SCOP sequence searches, alignments and genome assignments

Nucleic. Acids. Res.