Crystal structures of MTH1187 and its yeast ortholog YBL001c

As part of our structural genomics effort that focused on nonmembrane proteins in the proteome of the thermophilic archaeon Methanobacterium thermoautotrophicum (Mth),1 we selected the open reading frame (ORF) MTH1187. This protein of about 100 amino acid residues is conserved among roughly 25 bacterial and archaeal organisms in the current sequence database (PFAM01910.6) (Fig. 1), although its function in these organisms is unknown. In addition, MTH1187 shares 27% sequence identity with the ORF YBL001c in the yeast genome and is the only eukaryotic ortholog that can be identified based on the currently available sequences. The exact biochemical function of YBL001c is not known. However, inactivation of this gene, via transposon insertion, makes the yeast hypersensitive to an agent that perturbs cell surface polymers.2 This suggests that YBL001c may have a role in yeast cell-wall biogenesis, and this ORF is also known as ECM15 (extracellular mutant 15).2 Alignment of the sequences MTH1187, YBL001c, and others. Residues in the hydrophobic core of the monomer, dimer, and tetramer are shown in green, cyan, and purple, respectively. Residues in the sulfate-binding site are shown in red. The amino acid sequence numbers for MTH1187 and YBL001c are shown at the top and bottom, respectively. A dash represents a residue that is identical to that in MTH1187, whereas a dot represents a deletion; S.S., secondary structures. The crystal structures of MTH1187 and YBL001c have been determined at resolutions of 2.3 and 1.8 Å, respectively, and deposited at the Protein Data Bank (PDB) (Table I). As expected, based on their sequence conservation, the two proteins have similar structures overall. A total of 97 equivalent Cα atoms can be superimposed to within 3 Å of each other, and the root-mean-square distance (RMSD) for these atoms is 1.5 Å. Each monomer of the protein contains well-defined secondary structural elements, including a four-stranded antiparallel β-sheet (β1–β4) and three α-helices (αA–αC) [Fig. 2(a)]. Helix αC extends away from the rest of the structure [Fig. 2(a)]. Such a conformation is probably unstable for the molecule in the monomeric state, but these residues are stabilized by quaternary interactions in the tetramer, as well as by the binding of a sulfate ion [Fig. 2(a)]. Structure of MTH1187/YBL001c. (a) Schematic drawing of the monomer of MTH1187. The β-strands are shown in cyan, and the α-helices in yellow. The sulfate ion is shown as stick models. (b) The dimer of MTH1187, with the two monomers colored yellow and green, respectively. (c) The tetramer of MTH1187, with the monomers in yellow, green, cyan, and purple. (d) The tetramer of MTH1187 in a different view, showing the domain swapping of the αC helix. Produced with Ribbons.14 Structural searches of the PDB, with the programs PrISM,3 CE,4 and DALI,5 revealed that the monomer has a ferredoxin-like fold, with the exception of the αC helix in the tetramer interface. This ferredoxin-like fold is present in various proteins, including ribosomal protein S6, RNA- and DNA-binding proteins, proteins involved in effector-mediated allosteric regulation, and copper chaperone proteins. Interestingly, this folding motif of a four-stranded antiparallel β-sheet with two helices on one face is often observed to mediate protein oligomerization, producing dimers, trimers, and tetramers for a number of proteins. Aspartate kinase-chorismate mutase-TyrA (ACT) and regulation of amino acid metabolism (RAM) domains are two examples of protein modules with this backbone fold.6, 7 These domains function as dimers, mediating protein dimerization as well as ligand binding and allosteric regulation. However, the organization of the dimer and the location of the ligand-binding site are different in ACT and RAM domains.7 The dimer of RAM domains is formed by the face-to-face arrangement of the β-sheets of the two monomers, producing a β-sandwich. In comparison, the dimer of ACT domains is formed by the side-to-side arrangement of the β-sheets, producing an eight-stranded β-sheet. A conserved tetramer with an extensive interface is observed in the structures of MTH1187 and YBL001c [Fig. 2(c)], which suggests that these proteins are likely to exist as tetramers in solution as well. The dimer of MTH1187/YBL001c is formed by side-to-side arrangment of the monomers, producing an eight-stranded β-sheet [Fig. 2(b)]. However, this dimer formation is mediated by strand β4 of the monomer, whereas dimerization of the ACT domains is mediated by strand β2, at the other edge of the β-sheet [Fig. 2(a)]. The tetramer is formed by the face-to-face arrangement of the two dimers [Fig. 2(d)]. The αC helix participates in domain swapping in this tetramer interface [Fig. 2(d)]. Overall, MTH1187/YBL001c appears to represent a novel mode of oligomerization for this structurally conserved domain. In the structures of both MTH1187 and YBL001c, ordered sulfate ions associated with the tetramers are observed [Fig. 2(c)]. The ions are located at the dimer–dimer interfaces [Fig. 2(d)], which may provide additional stabilization of the tetramer. The sulfate-binding sites in the MTH1187 and YBL001c tetramers are located at structurally equivalent positions, and residues lining this binding site show enhanced conservation among these proteins (Fig. 1). An evolutionary analysis of the MTH1187/YBL001c family, performed with the program ConSurf (http://consurf.tau.ac.il/),8 indicates clustering of conserved residues to the sites of the bound sulfate, in addition to the core of the tetramer. This suggests that the observed binding site may have a role in the natural functions of these proteins, although the natural ligand(s) of this site is not known. Interestingly, both MTH1187 and YBL001c contain internal cavities of postive electrostatic potential immediately below the bound sulfate molecules. MTH1187 and YBL001c each contain eight ionizable residues buried in the central core of the tetramer, two contributed from each monomer, including Glu 5 and Lys 77 in MTH1187, and Asp 9 and Arg 80 in equivalent positions in YBL001c. These buried residues are involved in a network of hydrogen-bonding and ionic interactions. Interestingly, the sequences of most members of the MTH1187/YBL001c family conserve an acidic and basic residue at the positions Glu 5 and Lys 77 in MTH1187 (Fig. 1), suggesting that they may play a structural or function role, possibly in the assembly of the tetramer interface. Our structural analysis suggests that MTH1187/YBL001c is likely a protein–protein interaction module, and the function of this protein may be regulated by the binding of small-molecule ligands (possibly sulfate ions). Yeast two-hybrid screening with YBL001c identified four potential binding partners for this protein, YDR510w, YNL189w, YPL068c, and YER067w,9 although the biologic relevance of these putative interactions remains to be demonstrated. Of these proteins, YDR510w is a ubiquitin-like protein and may be involved in the structure or function of the eukaryotic kinetochore, and YNL189w is a homolog of karyopherin alpha. The functions of YPL068c and YER067w are currently not known. With our structural information, it would be interesting to assess the functional importance of the sulfate-binding site, and to identify its natural ligand(s). The MTH1187 and YBL001c genes were amplified by polymerase chain reaction (PCR) and cloned into the pET15b vector (Novagen), respectively. Recombinant proteins were expressed with a hexa-histidine fusion tag at the N-terminus in Escherichia coli BL21 (DE3). The cells were lysed in a buffer containing 50 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) (pH 7.5), 500 mM NaCl, 5% (v/v) glycerol, 1 mM dithiothreitol (DTT), 1 mM phenylmethanesulfonyl fluoride (PMSF), and 0.5 mM benzamidine. The soluble recombinant protein was bound to Ni+2-affinity resin and eluted in a buffer containing 50 mM HEPES (pH 7.5), 500 mM NaCl, 5% (v/v) glycerol, and 250 mM imidazole. The purified protein was dialyzed extensively against a buffer containing 10 mM HEPES and 500 mM NaCl, concentrated to 20 mg/mL, and stored at 4°C. The selenomethionine (Se-Met)-labeled protein was expressed in the methionine auxotroph E. coli strain B834 (DE3) (Novagen) and purified under the same conditions as the native protein, except that 5 mM β-mercaptoethanol was included in all buffers. The crystallization condition for MTH1187 consists of 0.1 M sodium acetate (pH 4.6), 2 M ammonium sulfate, and 14% glycerol, and that for YBL001c contains 0.1 M Tris (pH 8.5), 2 M ammonium sulfate, and 12% 2-methyl-2,4-pentanediol (MPD). X-ray diffraction data to 2.3 Å resolution for MTH1187, and to 1.8 Å resolution for YBL001c, were collected at the X4A beamline of the National Synchrotron Light Source (NSLS). The diffraction images were processed with the HKL package.10 The data-processing statistics are summarized in Table I. For both crystals, only a single-wavelength seleno-methionyl anomalous diffraction data set was collected.11 The positions of the Se atoms were determined based on the anomalous differences. After single-wavelength anomalous diffraction (SAD) phasing and solvent flattening, the atomic model of the protein was built into the electron density map with the program O.12 The structure refinement was carried out with the program CNS (Crystallography & NMR System).13 The refinement statistics are summarized in Table I. There are two fortuitous disulfide bonds in the MTH1187 tetramer, between Cys14 in one monomer and the equivalent Cys residue in another. Structural comparison with the YBL001c tetramer shows that they have little impact on the organization of the tetramer. Our thanks to Gerwald Jogl, Zhiru Yang, and Hailong Zhang for help with the data collection; to Craig Ogata for access to the X4A beamline at NSLS; to members of the Ontario Center for Structural Proteomics for help with protein purification and crystallization; and to Emil Alexov for helpful discussion.

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