Crystal structure of the YjgF/YER057c/UK114 family protein from the hyperthermophilic archaeon Sulfolobus tokodaii strain 7

• DOI: 10.1002/prot.20778
• Publication Date: 2005-12-01T22:48:54Z
• Authors: Takuya Miyakawa; Woo Cheol Lee; Ken‐ichi Hatano; Yusuke Kato; Yoriko Sawano; Ken‐ichi Miyazono; Koji Nagata; Masaru Tanokura
• Keywords: Sulfolobus; Strain (injury)

The YjgF/YER057c/UK114 family of proteins (referred to as the YjgF in this article) includes over 200 prokaryotic and eukaryotic consisting ca. 130 amino acid residues.1 protein members show diverse functions. In bacteria yeasts, they play prerequisite roles biosynthesis essential compounds such isoleucine,2-4 thiamine,2 purine.5 For example, gene disruption a yeast member that is involved isoleucine lethal cells.4 On other hand, mammalian members, whose expression upregulated upon cellular differentiation,6, 7 inhibit translations8, 9 by degrading mRNAs.10 Six crystal structures have been reported thus far for YjgF-family proteins. These are homotrimeric, with each subunit having β-barrel-like structure surrounded two α-helices.11-14 overall fold oligomeric state quite similar those chorismate mutase family.12 catalytic sites not clearly revealed, although three clefts between subunits presumed be analogy mutases.11, 12 fact, human hp14.5 can entrap small molecules benzoic within clefts.14 Thus far, no archaeal analyzed functionally or structurally. present study, we determined ST0811 from hyperthermophilic archaeon Sulfolobus tokodaii strain 715 compared bacterial counterparts. Our results indicate structurally more orthologs than ones. Protein expression, purification, crystallization were performed described.16 crystals obtained sitting-drop vapor-diffusion method at 293 K 3 days. reservoir solution used 16% (w/v) PEG 10,000, 0.1 M Bis-Tris (pH 5.3), ammonium acetate. Crystals transferred into cryoprotectant containing 18%(w/v) 10,000 precipitant, 0.12 acetate, 20% (v/v) glycerol then flash-cooled nitrogen stream. Diffraction data collected 100 an R-AXIS VII image plate detector mounted on Rigaku FR-E rotating-anode X-ray generator (Rigaku, Japan) using operation software CrystalClear (Rigaku/MSC) processed MOSFLM17 SCALA.18 was solved molecular replacement program MOLREP CCP4 suites19 coordinates Bacillus subtilis YabJ (PDB code 1QD9, 50% sequence identity ST0811).12 run resolution range 19.0–2.0 Å space group R3. Five percent reflections excluded total cross-validation Rfree value. rigid-body model initially refined Refmac5,20 several cycles manual rebuilding refinement XtalView21 Refmac5. Water automatically picked up ARP/wARP program,22 confirmed based peak heights distance criteria Fo-Fc 2Fo-Fc maps. quality evaluated PROCHECK.23 deposited Data Bank accession number 1X25. Structural analysis carried out following computer programs: Swiss-PdbViewer24 superposition molecules, calculation RMS deviations secondary structure-based alignment; CCP419 trimeric-contact surface; ESPript25 preparation alignment figure; PyMol (http://pymol.sourceforge.net) depiction structure; GRASP26 electrostatic potential surface. Other structural comparison B. 1QD9),12 Escherichia coli 1QU9),11 Yeo7 1JD1), goat UK114 1NQ3),13 rat L-PSP 1QAH), 1ONI).14 Dynamic light-scattering (DLS) measurements DynaPro (Protein solutions). prepared 5.0 mg/mL 10 mM Tris-HCl 7.1) measured 298 K. DLS Dynamics v5.1 solutions), 20 points baseline levels 0.998–1.002 calculate Stokes radius polydispersity ST0811. found belong rhombohedral R3, hexagonal unit-cell parameters = b 54.98 c 223.16 Å. Two contained per asymmetric unit (VM 2.3 Å3 Da−1, 47% solvent).27 2.0 resolution. final electron density allowed positioning all residues except N- C-terminal glycine chain A 210 water molecules. Rfactor 14.8 18.8%, respectively. Ramachandran plot,28 92.0% fell most favored regions, rest additionally regions. collection statistics summarized Table I. monomer shows α + β arrangement βββαβαββ referred mutase-like [Fig. 1(a)].11, β-sheet, order strands 1-2-3-6-4-5, where β4 β5 parallel antiparallel. forms homotrimeric axis 1(b)], do solved. β-sheets arranged manner six α-helices. contact area any 1890 Å2, 70% it occupied hydrophobic residues, which contributes stabilize trimer 1(b)]. exists homotrimer only but also solution, shown data. (27 Å) consistent maximum (28 Å). (a) Ribbon diagram monomeric α- 310-helices colored red β-strands blue. Side-chains represented stick models (magenta, β-strands; cyan, hydrophilic cavity; orange, cleft). (b) viewed cavity side (left) bottom (right). Each red, blue, green. Hydrophobic cluster, purple mesh, formed chains magenta (a). side-chains Thr25 Tyr27 protrude cluster cavity. positions allow heads. All fit 0.74–0.90 125 Cα positions. As Figure 2, differences come inserted gap α2 E. additional both C-termini orthologs. Thus, assembly would influence their diversity function. Structure-based sequential available structure. assignment indicated helices(α- 310-helices) arrows (β-strands). Residues forming cleft open circles (○) filled (•), Conserved boxed. Hydrophilic cavity, Tyr27, whereas charged around orthologs, Asp23 Arg24, stereo model. green, hydrogen bonds broken lines. Trimeric surface located subunits.12, 14 has clefts, × 13 11 size. conserved variable 2(a). Some Ser29, Gly30, Arg102, Glu117, connect scaffold 2(b)]. suggesting suitable binding benzoates, observed hp14.5.14 β3 β2-β3 turn. separated clusters β-sheets. well 2(a)], convey properties 3(a). Because does negatively Asp23, different 3(a)]. showing L-PSP. regions corresponding extended-loop Electrostatic ST0811, YjgF, Positive negative potentials blue thresholds ±10 kT/e, positive rather 3(b)]. reflective isoelectric calculated sequences L-PSP, YjgF: 8.5, 5.2, addition, trimeric stable presence strong (5% perchloric acid) monitored far-UV CD spectroscopy (data shown). This property histone proteins.6, 29 Unlike possess RNase activity.10, 30 positively these important attracting RNA able interact manner. Although experimental evidence must obtained, propose here archaea may bind regulate stability work supported part National Project Functional Analyses Ministry Education, Culture, Sports, Science Technology Japan Grants-in-Aid Scientific Research Japan.