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1.  Structure of the Rho-specific guanine nucleotide-exchange factor Xpln 
The Xpln crystal structure provides structural insights into Rho GTPase binding.
Xpln is a guanine nucleotide-exchange factor (GEF) for Rho GTPases. A Dbl homology (DH) domain followed by a pleckstrin homology (PH) domain is a widely adopted GEF-domain architecture. The Xpln structure solely comprises these two domains. Xpln activates RhoA and RhoB, but not RhoC, although their GTPase sequences are highly conserved. The molecular mechanism of the selectivity of Xpln for Rho GTPases is still unclear. In this study, the crystal structure of the tandemly arranged DH-PH domains of mouse Xpln, with a single molecule in the asymmetric unit, was determined at 1.79 Å resolution by the multiwavelength anomalous dispersion method. The DH-PH domains of Xpln share high structural similarity with those from neuroepithelial cell-transforming gene 1 protein, PDZ-RhoGEF, leukaemia-associated RhoGEF and intersectins 1 and 2. The crystal structure indicated that the α4–α5 loop in the DH domain is flexible and that the DH and PH domains interact with each other intramolecularly, thus suggesting that PH-domain rearrangement occurs upon RhoA binding.
doi:10.1107/S1744309112045265
PMCID: PMC3509964  PMID: 23192023
GEF proteins; DH-PH module structure
2.  Crystallization and preliminary X-ray crystallographic analysis of Aquifex aeolicus SelA, a bacterial selenocysteine synthase 
The bacterial selenocysteine synthase SelA from Aquifex aeolicus was crystallized and the diffraction resolution was improved by lysine-residue methylation, truncation of N-terminal region (ΔN), and Lys-to-Ala point mutations. Phases were determined by using a selenomethionine-substituted crystal of the ΔN mutant.
Selenocysteine (Sec), the 21st amino acid, is synthesized on its specific tRNA (tRNASec) via a multi-step process. In bacteria, tRNASec is ligated first with serine by seryl-tRNA synthetase, which is followed by Ser-to-Sec conversion by Sec synthase (SelA). To elucidate its structure and catalytic mechanism, Aquifex aeolicus SelA was crystallized. Although wild-type SelA crystals diffracted X-­rays poorly (to up to 8 Å resolution), the resolution was improved by introducing a quadruple point mutation targeting the loop regions and by methylating the lysine residues, which yielded 3.9 Å resolution diffraction data from a full-length SelA crystal. Truncation of the N-terminal region (ΔN) also improved the resolution. A 3.3 Å resolution data set for phase determination was obtained from a crystal of selenomethionine-substituted Lys-methylated SelA-ΔN.
doi:10.1107/S1744309112033519
PMCID: PMC3433215  PMID: 22949212
SelA; Aquifex aeolicus; selenocysteine synthase
3.  Flexibility of the P-loop of Pim-1 kinase: observation of a novel conformation induced by interaction with an inhibitor 
The structures of Pim1 kinase in complex with in silico screening hits and several subsequently optimized inhibitors, are reported.
The serine/threonine kinase Pim-1 is emerging as a promising target for cancer therapeutics. Much attention has recently been focused on identifying potential Pim-1 inhibitor candidates for the treatment of haematopoietic malignancies. The outcome of a rational drug-design project has recently been reported [Nakano et al. (2012 ▶), J. Med. Chem. 55, 5151–5156]. The report described the process of optimization of the structure–activity relationship and detailed from a medicinal chemistry perspective the development of a low-potency and nonselective compound initially identified from in silico screening into a potent, selective and metabolically stable Pim-1 inhibitor. Here, the structures of the initial in silico hits are reported and the noteworthy features of the Pim-1 complex structures are described. A particular focus was placed on the rearrangement of the glycine-rich P-loop region that was observed for one of the initial compounds, (Z)-7-(azepan-1-ylmethyl)-2-[(1H-indol-3-­yl)methylidene]-6-hydroxy-1-benzofuran-3(2H)-one (compound 1), and was also found in all further derivatives. This novel P-loop conformation, which appears to be stabilized by an additional interaction with the β3 strand located above the binding site, is not usually observed in Pim-1 structures.
doi:10.1107/S1744309112027108
PMCID: PMC3412761  PMID: 22869110
Pim-1; kinases; inhibitors
4.  Crystallization and preliminary X-ray crystallographic analysis of bacterial tRNASec in complex with seryl-tRNA synthetase 
Bacterial selenocysteine tRNA was crystallized as the heterologous complex with archaeal seryl-tRNA synthetase. X-ray diffraction was improved by introducing point mutations and heavy-atom labeling, and a 3.2 Å diffraction data set for phase determination was obtained from a platinum-labeled crystal.
Selenocysteine (Sec) is translationally incorporated into proteins in response to the UGA codon. The tRNA specific to Sec (tRNASec) is first ligated with serine by seryl-tRNA synthetase (SerRS). To elucidate the tertiary structure of bacterial tRNASec and its specific interaction with SerRS, the bacterial tRNASec from Aquifex aeolicus was crystallized as the heterologous complex with the archaeal SerRS from Methanopyrus kandleri. Although X-ray diffraction by crystals of tRNASec in complex with wild-type SerRS was rather poor (to 5.7 Å resolution), the resolution was improved by introducing point mutations targeting the crystal-packing interface. Heavy-atom labelling also contributed to resolution improvement. A 3.2 Å resolution diffraction data set for phase determination was obtained from a K2Pt(CN)4-soaked crystal.
doi:10.1107/S1744309112016004
PMCID: PMC3370909  PMID: 22684069
seryl-tRNA synthetase; tRNASec
5.  Structure of the hypothetical DUF1811-family protein GK0453 from Geobacillus kaustophilus HTA426 
The gene encoding the hypothetical DUF1811-family protein GK0453 from G. kaustophilus was cloned and expressed. The crystal structure of the protein was determined by the molecular-replacement method and was refined to 2.2 Å resolution.
The crystal structure of a conserved hypothetical protein, GK0453, from Geobacillus kaustophilus has been determined to 2.2 Å resolution. The crystal belonged to space group P43212, with unit-cell parameters a = b = 75.69, c = 64.18 Å. The structure was determined by the molecular-replacement method and was refined to a final R factor of 22.6% (R free = 26.3%). Based on structural homology, the GK0453 protein possesses two independent binding sites and hence it may simultaneously interact with two proteins or with a protein and a nucleic acid.
doi:10.1107/S1744309113003369
PMCID: PMC3614154  PMID: 23545635
DUF1811; Geobacillus kaustophilus; helix–turn–helix motif; β-barrel domain
6.  Structure of the minimized α/β-hydrolase fold protein from Thermus thermophilus HB8 
Acta Crystallographica Section F  2007;63(Pt 12):993-997.
The crystal structure of the minimized α/β-hydrolase fold protein encoded by the gene TTHA1544 from T. thermophilus HB8 has been determined at 2.0 Å resolution.
The gene encoding TTHA1544 is a singleton found in the Thermus thermophilus HB8 genome and encodes a 131-amino-acid protein. The crystal structure of TTHA1544 has been determined at 2.0 Å resolution by the single-wavelength anomalous dispersion method in order to elucidate its function. There are two molecules in the asymmetric unit. Each molecule consists of four α-helices and six β-strands, with the β-strands composing a central β-sheet. A structural homology search revealed that the overall structure of TTHA1544 resembles the α/β-hydrolase fold, although TTHA1544 lacks the catalytic residues of a hydrolase. These results suggest that TTHA1544 represents the minimized α/β-hydrolase fold and that an additional component would be required for its activity.
doi:10.1107/S1744309107061106
PMCID: PMC2344104  PMID: 18084077
T. thermophilus HB8; TTHA1544; α/β-hydrolase fold; singleton
7.  Structures of a putative RNA 5-methyluridine methyltransferase, Thermus thermophilus TTHA1280, and its complex with S-adenosyl-l-homocysteine 
Acta Crystallographica Section F  2005;61(Pt 10):867-874.
Three structures of a putative RNA 5-methyluridine methyltransferase from T. thermophilus, including its complex with S-adenosyl-l-homocysteine, are presented. The structures reveal the mode of cofactor binding, architecture of the putative active site, and the presence of a deep cleft adjacent to the active site that may bind RNA.
The Thermus thermophilus hypothetical protein TTHA1280 belongs to a family of predicted S-adenosyl-l-methionine (AdoMet) dependent RNA methyltransferases (MTases) present in many bacterial and archaeal species. Inspection of amino-acid sequence motifs common to class I Rossmann-fold-like MTases suggested a specific role as an RNA 5-methyluridine MTase. Selenomethionine (SeMet) labelled and native versions of the protein were expressed, purified and crystallized. Two crystal forms of the SeMet-labelled apoprotein were obtained: SeMet-ApoI and SeMet-ApoII. Cocrystallization of the native protein with S-­adenosyl-l-homocysteine (AdoHcy) yielded a third crystal form, Native-AdoHcy. The SeMet-ApoI structure was solved by the multiple anomalous dispersion method and refined at 2.55 Å resolution. The SeMet-ApoII and Native-AdoHcy structures were solved by molecular replacement and refined at 1.80 and 2.60 Å, respectively. TTHA1280 formed a homodimer in the crystals and in solution. Each subunit folds into a three-domain structure composed of a small N-terminal PUA domain, a central α/β-domain and a C-terminal Rossmann-fold-like MTase domain. The three domains form an overall clamp-like shape, with the putative active site facing a deep cleft. The architecture of the active site is consistent with specific recognition of uridine and catalysis of methyl transfer to the 5-carbon position. The cleft is suitable in size and charge distribution for binding single-stranded RNA.
doi:10.1107/S1744309105029842
PMCID: PMC1991318  PMID: 16511182
PUA domain; RNA-modification enzyme; 5-methyluridine methyltransferase; S-adenosyl-l-homocysteine
8.  Overexpression, purification, crystallization and preliminary crystallographic studies of a hyperthermophilic adenylosuccinate synthetase from Pyrococcus horikoshii OT3 
A hyperthermophilic adenylosuccinate synthetase from P. horikoshii OT3, which is 90–120 amino acids shorter than those from the vast majority of organisms, was expressed, purified and crystallized and X-ray diffraction data were collected to 2.5 Å resolution.
Adenylosuccinate synthetase (AdSS) is a ubiquitous enzyme that catalyzes the first committed step in the conversion of inosine monophosphate (IMP) to adenosine monophosphate (AMP) in the purine-biosynthetic pathway. Although AdSS from the vast majority of organisms is 430–457 amino acids in length, AdSS sequences isolated from thermophilic archaea are 90–120 amino acids shorter. In this study, crystallographic studies of a short AdSS sequence from Pyrococcus horikoshii OT3 (PhAdSS) were performed in order to reveal the unusual structure of AdSS from thermophilic archaea. Crystals of PhAdSS were obtained by the microbatch-under-oil method and X-ray diffraction data were collected to 2.50 Å resolution. The crystal belonged to the trigonal space group P3212, with unit-cell parameters a = b = 57.2, c = 107.9 Å. There was one molecule per asymmetric unit, giving a Matthews coefficient of 2.17 Å3 Da−1 and an approximate solvent content of 43%. In contrast, the results of native polyacrylamide gel electrophoresis and analytical ultracentrifugation showed that the recombinant PhAdSS formed a dimer in solution.
doi:10.1107/S174430911104108X
PMCID: PMC3232137  PMID: 22139164
adenylosuccinate synthetases; purine-biosynthetic pathway; Pyrococcus horikoshii OT3
9.  Crystal structures, dynamics and functional implications of molybdenum-cofactor biosynthesis protein MogA from two thermophilic organisms 
Three crystal structures of the molybdenum-cofactor biosynthesis protein MogA from two highly thermophilic organisms have been determined at high resolution. Comparative analyses revealed the residues involved in oligomerization. In addition, molecular-dynamics and docking studies suggested the binding affinities of several small molecules towards MogA and homologous proteins.
Molybdenum-cofactor (Moco) biosynthesis is an evolutionarily conserved pathway in almost all kingdoms of life, including humans. Two proteins, MogA and MoeA, catalyze the last step of this pathway in bacteria, whereas a single two-domain protein carries out catalysis in eukaryotes. Here, three crystal structures of the Moco-biosynthesis protein MogA from the two thermophilic organisms Thermus thermophilus (TtMogA; 1.64 Å resolution, space group P21) and Aquifex aeolicus (AaMogA; 1.70 Å resolution, space group P21 and 1.90 Å resolution, space group P1) have been determined. The functional roles and the residues involved in oligomerization of the protein molecules have been identified based on a comparative analysis of these structures with those of homologous proteins. Furthermore, functional roles have been proposed for the N- and C-terminal residues. In addition, a possible protein–protein complex of MogA and MoeA has been proposed and the residues involved in protein–protein interactions are discussed. Several invariant water molecules and those present at the subunit interfaces have been identified and their possible structural and/or functional roles are described in brief. In addition, molecular-dynamics and docking studies with several small molecules (including the substrate and the product) have been carried out in order to estimate their binding affinities towards AaMogA and TtMogA. The results obtained are further compared with those obtained for homologous eukaryotic proteins.
doi:10.1107/S1744309110035037
PMCID: PMC3079962  PMID: 21206014
MogA; molybdenum-cofactor biosynthesis proteins
10.  Crystallization and preliminary X-ray crystallographic study of GenX, a lysyl-tRNA synthetase paralogue from Escherichia coli, in complex with translation elongation factor P 
GenX, a lysyl-tRNA synthetase paralogue from Escherichia coli, has been overexpressed in E. coli, purified by three chromatographic steps and cocrystallized with a lysyl adenylate analogue (LysAMS) by the hanging-drop vapour-diffusion method using PEG 4000 as a precipitant.
GenX, a lysyl-tRNA synthetase paralogue from Escherichia coli, was overexpressed in E. coli, purified by three chromatographic steps and cocrystallized with a lysyl adenylate analogue (LysAMS) by the hanging-drop vapour-diffusion method using PEG 4000 as a precipitant. The GenX–LysAMS crystals belonged to the triclinic space group P1, with unit-cell parameters a = 54.80, b = 69.15, c = 94.08 Å, α = 95.47, β = 106.51, γ = 90.46°, and diffracted to 1.9 Å resolution. Furthermore, GenX was cocrystallized with translation elongation factor P (EF-­P), which is believed to be a putative substrate of GenX, and LysAMS using PEG 4000 and ammonium sulfate as precipitants. The GenX–EF-P–LysAMS crystals belonged to the monoclinic space group P21, with unit-cell parameters a = 105.93, b = 102.96, c = 119.94 Å, β = 99.4°, and diffracted to 2.5 Å resolution. Structure determination of the E. coli GenX–LysAMS and GenX–EF-P–LysAMS complexes by molecular replacement was successful and structure refinements are now in progress.
doi:10.1107/S1744309110032008
PMCID: PMC2935242  PMID: 20823541
GenX; lysyl-tRNA synthetase paralogue; translation elongation factor P
11.  Cloning, expression, purification, crystallization and preliminary X-ray crystallographic study of the putative SAICAR synthetase (PH0239) from Pyrococcus horikoshii OT3 
SAICAR synthetase from P. horikoshii OT3 has been cloned, expressed, purified and crystallized.
The study of proteins involved in de novo biosynthesis of purine nucleotides is central in the development of antibiotics and anticancer drugs. In view of this, a protein from the hyperthermophile Pyrococcus horikoshii OT3 was isolated, purified and crystallized using the microbatch method. Its primary structure was found to be similar to that of SAICAR synthetase, which catalyses the seventh step of de novo purine biosynthesis. A diffraction-quality crystal was obtained using Hampton Research Crystal Screen II condition No. 34, consisting of 0.05 M cadmium sulfate hydrate, 0.1 M HEPES buffer pH 7.5 and 1.0 M sodium acetate trihydrate, with 40%(v/v) 1,4-butanediol as an additive. The crystal belonged to space group P31, with unit-cell parameters a = b = 95.62, c = 149.13 Å. Assuming the presence of a hexamer in the asymmetric unit resulted in a Matthews coefficient (V M) of 2.3 Å3 Da−1, corresponding to a solvent content of about 46%. A detailed study of this protein will yield insights into structural stability at high temperatures and should be highly relevant to the development of antibiotics and anticancer drugs targeting the biosynthesis of purine nucleotides.
doi:10.1107/S1744309109052026
PMCID: PMC2815687  PMID: 20124717
SAICAR synthetase; PH0239; Pyrococcus horikoshii OT3; purine biosynthesis
12.  Expression, purification and X-ray analysis of 1,3-­propanediol dehydrogenase (Aq_1145) from Aquifex aeolicus VF5 
1,3-Propanediol dehydrogenase (Aq_1145) from A. aeolicus VF5 has been overexpressed, purified and crystallized. The crystals diffracted to 2.4 Å resolution.
1,3-Propanediol dehydrogenase is an enzyme that catalyzes the oxidation of 1,3-­propanediol to 3-hydroxypropanal with the simultaneous reduction of NADP+ to NADPH. SeMet-labelled 1,3-propanediol dehydrogenase protein from the hyperthermophilic bacterium Aquifex aeolicus VF5 was overexpressed in Escherichia coli and purified to homogeneity. Crystals of this protein were grown from an acidic buffer with ammonium sulfate as the precipitant. Single-wavelength data were collected at the selenium peak to a resolution of 2.4 Å. The crystal belonged to space group P32, with unit-cell parameters a = b = 142.19, c = 123.34 Å. The structure contained two dimers in the asymmetric unit and was solved by the MR-SAD approach.
doi:10.1107/S1744309109052403
PMCID: PMC2815688  PMID: 20124718
1,3-propanediol dehydrogenase; Aquifex aeolicus VF5; Aq_1145
13.  Crystallization and preliminary X-ray crystallographic analysis of Thermus thermophilus transcription elongation complex bound to Gfh1 
To elucidate which RNA polymerase structural state a particular T. thermophilus Gre-family protein (Gfh1) associates with, the T. thermophilus RNAP elongation complex was cocrystallized with Gfh1.
RNA polymerase (RNAP) elongates RNA by iterative nucleotide-addition cycles (NAC). A specific structural state (or states) of RNAP may be the target of transcription elongation factors. Gfh1, a Thermus thermophilus Gre-family protein, inhibits NAC. To elucidate which RNAP structural state Gfh1 associates with, the T. thermophilus RNAP elongation complex (EC) was cocrystallized with Gfh1. Of the 70 DNA/RNA scaffolds tested, two (for EC1 and EC2) were successfully crystallized. In the presence of Gfh1, EC1 and EC2 yielded crystals belonging to space group P21 with similar unit-cell parameters (crystals 1 and 2, respectively). X-ray diffraction data sets were obtained at 3.6 and 3.8 Å resolution, respectively.
doi:10.1107/S1744309109049215
PMCID: PMC2805540  PMID: 20057074
GreA; nucleotide-addition cycle; transcript cleavage
14.  Structure of hypothetical Mo-cofactor biosynthesis protein B (ST2315) from Sulfolobus tokodaii  
The structure of a protein involved in the molybdopterin and molybdenum co-factor biosynthesis pathways of Sulfolobus tokodaii has been solved to a resolution of 1.9 Å.
The structure of a probable Mo-cofactor biosynthesis protein B from Sulfolobus tokodaii, belonging to space group P6422 with unit-cell parameters a = b = 136.68, c = 210.52 Å, was solved by molecular replacement to a resolution of 1.9 Å and refined to an R factor and R free of 16.8% and 18.5%, respectively. The asymmetric unit contains a trimer, while the biologically significant oligomer is predicted to be a hexamer by size-exclusion chromatography. The subunit structure and fold of ST2315 are similar to those of other enzymes that are known to be involved in the molybdopterin- and molybdenum cofactor-biosynthesis pathways.
doi:10.1107/S1744309109043772
PMCID: PMC2802863  PMID: 20054111
ST2315; Sulfolobus tokodaii; Mo-cofactor biosynthesis protein B
15.  Structure of SurE protein from Aquifex aeolicus VF5 at 1.5 Å resolution 
The structure of the stationary phase survival protein SurE protein from the hyperthermophile Aquifex aeolicus has been solved to 1.5 Å resolution. The divalent-metal-ion-dependent phosphatase active-site pocket is occupied by sulfate ions from the crystallization medium.
SurE is a stationary-phase survival protein found in bacteria, eukaryotes and archaea that exhibits a divalent-metal-ion-dependent phosphatase activity and acts as a nucleotidase and polyphosphate phosphohydrolase. The structure of the SurE protein from the hyperthermophile Aquifex aeolicus has been solved at 1.5 Å resolution using molecular replacement with one dimer in the asymmetric unit and refined to an R factor of 15.6%. The crystal packing reveals that two dimers assemble to form a tetramer, although gel-filtration chromatography showed the presence of only a dimer in solution. The phosphatase active-site pocket was occupied by sulfate ions from the crystallization medium.
doi:10.1107/S1744309109043814
PMCID: PMC2802864  PMID: 20054112
SurE; Aquifex aeolicus; stationary-phase survival
16.  Structure of d-lactate dehydrogenase from Aquifex aeolicus complexed with NAD+ and lactic acid (or pyruvate) 
The structure of d-lactate dehydrogenase from Aquifex aeolicus has been determined with each subunit of the homodimer in a ‘closed’ conformation and with the NAD+ cofactor and lactate (or pyruvate) bound at the inter-domain active-site cleft.
The crystal structure of d-lactate dehydrogenase from Aquifex aeolicus (aq_727) was determined to 2.12 Å resolution in space group P212121, with unit-cell parameters a = 90.94, b = 94.43, c = 188.85 Å. The structure was solved by molecular replacement using the coenzyme-binding domain of Lactobacillus helveticus d-lactate dehydrogenase and contained two homodimers in the asymmetric unit. Each subunit of the homodimer was found to be in a ‘closed’ conformation with the NADH cofactor bound to the coenzyme-binding domain and with a lactate (or pyruvate) molecule bound at the interdomain active-site cleft.
doi:10.1107/S1744309109044935
PMCID: PMC2802865  PMID: 20054113
d-lactate dehydrogenase; Aquifex aeolicus
17.  The structure of an archaeal ribose-5-phosphate isomerase from Methanocaldococcus jannaschii (MJ1603) 
The structure of ribose-5-phosphate isomerase from Methanocaldococcus jannaschii has been solved to 1.78 Å resolution, with the active site occupied by two molecules of propylene glycol mimicking the binding of a known arabinose-5-phosphate inhibitor.
Ribose-5-phosphate isomerase is a ubiquitous intracellular enzyme of bacterial, plant and animal origin that is involved in the pentose phosphate cycle, an essential component of cellular carbohydrate metabolism. Specifically, the enzyme catalyses the reversible conversion of ribose 5-phosphate to ribulose 5-­phosphate. The structure of ribose-5-phosphate isomerase from Methano­caldococcus jannaschii has been solved in space group P21 to 1.78 Å resolution using molecular replacement with one homotetramer in the asymmetric unit and refined to an R factor of 14.8%. The active site in each subunit was occupied by two molecules of propylene glycol in different orientations, one of which corresponds to the location of the phosphate moiety and the other to the location of the furanose ring of the inhibitor.
doi:10.1107/S1744309109044923
PMCID: PMC2802866  PMID: 20054114
ribose-5-phosphate isomerase; Methanocaldococcus jannaschii; pentose phosphate cycle
18.  Structure of a putative β-phosphoglucomutase (TM1254) from Thermotoga maritima  
The structure of a putative β-phosphoglucomutase from Thermotoga maritima belonging to the haloacid dehalogenase (HAD) hydrolase family has been determined to 1.74 Å resolution.
The structure of TM1254, a putative β-phosphoglucomutase from T. maritima, was determined to 1.74 Å resolution in a high-throughput structural genomics programme. Diffraction data were obtained from crystals belonging to space group P22121, with unit-cell parameters a = 48.16, b = 66.70, c = 83.80 Å, and were refined to an R factor of 19.2%. The asymmetric unit contained one protein molecule which is comprised of two domains. Structural homologues were found from protein databases that confirmed a strong resemblance between TM1254 and members of the haloacid dehalogenase (HAD) hydrolase family.
doi:10.1107/S1744309109046302
PMCID: PMC2802867  PMID: 20054115
β-phosphoglucomutase; Thermotoga maritima
19.  Structure of dihydrodipicolinate synthase from Methanocaldococcus jannaschii  
The crystal structure of dihydrodipicolinate synthase from the (S)-lysine synthesis pathway of Methanocaldococcus jannaschii has been solved to 2.2 Å resolution, revealing a functional homotetramer.
In bacteria and plants, dihydrodipicolinate synthase (DHDPS) plays a key role in the (S)-lysine biosynthesis pathway. DHDPS catalyzes the first step of the condensation of (S)-aspartate-β-semialdehyde and pyruvate to form an unstable compound, (4S)-4-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinic acid. The activity of DHDPS is allosterically regulated by (S)-lysine, a feedback inhibitor. The crystal structure of DHDPS from Methanocaldococcus jannaschii (MjDHDPS) was solved by the molecular-replacement method and was refined to 2.2 Å resolution. The structure revealed that MjDHDPS forms a functional homo­tetramer, as also observed in Escherichia coli DHDPS, Thermotoga maritima DHDPS and Bacillus anthracis DHDPS. The binding-site region of MjDHDPS is essentially similar to those found in other known DHDPS structures.
doi:10.1107/S174430910904651X
PMCID: PMC2802868  PMID: 20054116
dihydrodipicolinate synthase; Methanocaldococcus jannaschii
20.  Structure of putative 4-amino-4-deoxychorismate lyase from Thermus thermophilus HB8 
The putative 4-amino-4-deoxychorismate lyase (TTHA0621) from T. thermophilus HB8 was cloned, overexpressed, purified and crystallized. Its crystal structure was determined by a combination of SAD and molecular-replacement methods and was refined to 1.93 Å resolution.
The pyridoxal 5′-phosphate-dependent enzyme 4-amino-4-deoxychorismate lyase converts 4-amino-4-deoxychorismate to p-aminobenzoate and pyruvate in one of the crucial steps in the folate-biosynthesis pathway. The primary structure of the hypothetical protein TTHA0621 from Thermus thermophilus HB8 suggests that TTHA0621 is a putative 4-amino-4-deoxychorismate lyase. Here, the crystal structure of TTHA0621 is reported at 1.93 Å resolution. The asymmetric unit contained four NCS molecules related by 222 noncrystallographic symmetry, in which the formation of intact dimers may be functionally important. The cofactor pyridoxal 5′-phosphate (PLP) binds to the protein in the large cleft formed by the N-terminal and C-terminal domains of TTHA0621. The high structural similarity and the conservation of the functional residues in the catalytic region compared with 4-amino-4-deoxychorismate lyase (PabC; EC 4.1.3.38) from Escherichia coli suggest that the TTHA0621 protein may also possess 4-amino-4-deoxychorismate lyase activity.
doi:10.1107/S1744309109050052
PMCID: PMC2802870  PMID: 20054118
4-amino-4-deoxychorismate lyase; pyridoxal 5′-phosphate; folate biosynthesis; Thermus thermophilus HB8
21.  Structure of glyceraldehyde-3-phosphate dehydrogenase from the archaeal hyperthermophile Methanocaldococcus jannaschii  
The structure of glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaeon Methanocaldococcus jannaschii was determined to 1.81 Å resolution with the NADP+ cofactor at the nucleotide binding site.
The X-ray crystal structure of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the hyperthermophilic archaeon Methanocaldococcus jannaschii (Mj-GAPDH) was determined to 1.81 Å resolution. The crystal belonged to space group C2221, with unit-cell parameters a = 83.4, b = 152.0, c = 118.6 Å. The structure was solved by molecular replacement and was refined to a final R factor of 17.1% (R free = 19.8%). The final structure included the cofactor NADP+ at the nucleotide-binding site and featured unoccupied inorganic and substrate phosphate-binding sites. A comparison with GAPDH structures from mesophilic sources suggested that Mj-GAPDH is stabilized by extensive electrostatic interactions between the C-terminal α-helices and various distal loop regions, which are likely to contribute to thermal stability. The key phosphate-binding residues in the active site of Mj-GAPDH are conserved in other archaeal GAPDH proteins. These residues undergo a conformational shift in response to occupancy of the inorganic phosphate site.
doi:10.1107/S1744309109047046
PMCID: PMC2802869  PMID: 20054117
GAPDH; Methanocaldococcus jannaschii; glycolysis; gluconeogenesis; hyperthermophiles
22.  New structural insights and molecular-modelling studies of 4-methyl-5-β-hydroxyethylthiazole kinase from Pyrococcus horikoshii OT3 (PhThiK) 
The first crystal structure of 4-methyl-5-β-hydroxyethylthiazole kinase from an archaeon (P. horikoshii OT3) has been determined at 1.85 Å resolution. Comparative analyses of sequences and structures and modelling studies are presented.
4-Methyl-5-β-hydroxyethylthiazole kinase (ThiK) catalyses the phosphorylation of the hydroxyl group of 4-methyl-5-β-hydroxyethylthiazole. This work reports the first crystal structure of an archaeal ThiK: that from Pyrococcus horikoshii OT3 (PhThiK) at 1.85 Å resolution with a phosphate ion occupying the position of the β-phosphate of the nucleotide. The topology of this enzyme shows the typical ribokinase fold of an α/β protein. The overall structure of PhThiK is similar to those of Bacillus subtilis ThiK (BsThiK) and Enterococcus faecalis V583 ThiK (EfThiK). Sequence analysis of ThiK enzymes from various sources indicated that three-quarters of the residues involved in interfacial regions are conserved. It also revealed that the amino-acid residues in the nucleotide-binding, magnesium ion-binding and substrate-binding sites are conserved. Binding of the nucleotide and substrate to the ThiK enzyme do not influence the quaternary association (trimer) as revealed by the crystal structure of PhThiK.
doi:10.1107/S1744309109036033
PMCID: PMC2765881  PMID: 19851002
4-methyl-5-β-hydroxyethylthiazole kinase; Pyrococcus horikoshii OT3; molecular modelling
23.  Purification, crystallization and preliminary X-ray diffraction analysis of the putative ABC transporter ATP-binding protein from Thermotoga maritima  
The putative ABC transporter ATP-binding protein TM0222 from T. maritima was cloned, overproduced, purified and crystallized. A complete MAD diffraction data set has been collected to 2.3 Å resolution.
Adenosine triphosphate (ATP) binding cassette transporters (ABC transporters) are ATP hydrolysis-dependent transmembrane transporters. Here, the overproduction, purification and crystallization of the putative ABC transporter ATP-binding protein TM0222 from Thermotoga maritima are reported. The protein was crystallized in the hexagonal space group P6422, with unit-cell parameters a = b = 148.49, c = 106.96 Å, γ = 120.0°. Assuming the presence of two molecules in the asymmetric unit, the calculated V M is 2.84 Å3 Da−1, which corresponds to a solvent content of 56.6%. A three-wavelength MAD data set was collected to 2.3 Å resolution from SeMet-substituted TM0222 crystals. Data sets were collected on the BL38B1 beamline at SPring-8, Japan.
doi:10.1107/S1744309108013778
PMCID: PMC2496867  PMID: 18540059
ATP-binding proteins; transmembrane transporters; Thermotoga maritima; MAD data
24.  Structure of an N-terminally truncated selenophosphate synthetase from Aquifex aeolicus  
The crystal structure of an N-terminally (25 residues) truncated fragment of selenophosphate synthetase (SPS-ΔN) from Aquifex aeolicus has been determined at 2.0 Å resolution.
Selenophosphate synthetase (SPS) catalyzes the activation of selenide with ATP to synthesize selenophosphate, the reactive selenium donor for biosyntheses of both the 21st amino acid selenocysteine and 2-selenouridine nucleotides in tRNA anticodons. The crystal structure of an N-terminally (25 residues) truncated fragment of SPS (SPS-ΔN) from Aquifex aeolicus has been determined at 2.0 Å resolution. The structure revealed SPS to be a two-domain α/β protein, with domain folds that are homologous to those of PurM-superfamily proteins. In the crystal, six monomers of SPS-ΔN form a hexamer of 204 kDa, whereas the molecular weight estimated by ultracentrifugation was ∼63 kDa, which is comparable to the calculated weight of the dimer (68 kDa).
doi:10.1107/S1744309108012074
PMCID: PMC2496870  PMID: 18540050
SelD; selenocysteine; selenophosphate; selenouridine
25.  Crystallization screening test for the whole-cell project on Thermus thermophilus HB8 
Three conventional robots were subjected to a crystallization screening test involving 18 proteins from T. thermophilus HB8 using the sitting- and hanging-drop vapour-diffusion and microbatch methods. The number of diffraction-quality crystals and the amount of time required to obtain visible crystals depended greatly on the robots used. The combined use of different robots, especially for protein samples exhibiting low crystallization success rates, significantly increased the chance of obtaining diffraction-quality crystals.
It was essential for the structural genomics of Thermus thermophilus HB8 to efficiently crystallize a number of proteins. To this end, three conventional robots, an HTS-80 (sitting-drop vapour diffusion), a Crystal Finder (hanging-drop vapour diffusion) and a TERA (modified microbatch) robot, were subjected to a crystallization condition screening test involving 18 proteins from T. thermophilus HB8. In addition, a TOPAZ (microfluidic free-interface diffusion) designed specifically for initial screening was also briefly examined. The number of diffraction-quality crystals and the time of appearance of crystals increased in the order HTS-80, Crystal Finder, TERA. With the HTS-80 and Crystal Finder, the time of appearance was short and the rate of salt crystallization was low. With the TERA, the number of diffraction-quality crystals was high, while the time of appearance was long and the rate of salt crystallization was relatively high. For the protein samples exhibiting low crystallization success rates, there were few crystallization conditions that were common to the robots used. In some cases, the success rate depended greatly on the robot used. The TOPAZ showed the shortest time of appearance and the highest success rate, although the crystals obtained were too small for diffraction studies. These results showed that the combined use of different robots significantly increases the chance of obtaining crystals, especially for proteins exhibiting low crystallization success rates. The structures of 360 of 944 purified proteins have been successfully determined through the combined use of an HTS-80 and a TERA.
doi:10.1107/S1744309108013572
PMCID: PMC2496871  PMID: 18540056
protein crystallization; crystallization screening; crystallization success rates

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