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.
PUA domain; RNA-modification enzyme; 5-methyluridine methyltransferase; S-adenosyl-l-homocysteine
Alanine and glutamine mutations were made to the same 15 lysine positions on the surface of E. coli malate synthase G and the impact on crystallization observed. The results support lysine replacement for improvement of crystallization and provide insight into site selection and type of amino-acid replacement.
Two proposals recommend substitution of surface lysine residues as a means to improve the quality of protein crystals. In proposal I, substitution of lysine by alanine has been suggested to improve crystallization by reducing the entropic cost of ordering flexible side chains at crystal contacts. In proposal II, substitution of lysine by residues more commonly found in crystal contacts, such as glutamine, has been proposed to improve crystallization. 15 lysine residues on the surface of Escherichia coli malate synthase G, distributed over a variety of secondary structures, were individually mutated to both alanine and glutamine. For 28 variants, detailed studies of the effect on enzymatic activity and crystallization were conducted. This has permitted direct comparison of the relative effects of the two types of mutations. While none of the variants produced crystals suitable for X-ray structural determination, small crystals were obtained in a wide variety of conditions, in support of the general approach. Glutamine substitutions were found to be more effective than alanine in producing crystals, in support of proposal II. Secondary structure at the site of mutation does not appear to play a major role in determining the rate of success.
malate synthase; protein engineering; mutagenesis
The GreB transcription cleavage factor of E. coli and the Gfh1 transcription elongation factor of T. thermophilus were cloned, expressed, purified and crystallized. Complete diffraction data sets were collected for the GreB and Gfh1 crystals to 2.6 and 2.8 Å resolution, respectively. Structure determination of these proteins is now in progress.
The Escherichia coli gene encoding the transcription cleavage factor GreB and the Thermus thermophilus gene encoding the anti-GreA transcription factor Gfh1 were cloned and expressed and the purified proteins were crystallized by the sitting-drop vapor-diffusion technique. The GreB and Gfh1 crystals, which were improved by macroseeding, belong to space group P41212 (or P43212), with unit-cell parameters a = b = 148, c = 115.2 Å and a = b = 59.3, c = 218.9 Å, respectively. Complete diffraction data sets were collected for the GreB and Gfh1 crystals to 2.6 and 2.8 Å resolution, respectively. Crystals of the selenomethionine proteins were obtained by microseeding using the native protein crystals and diffract as well as the native ones. The structure determination of these proteins is now in progress.
transcription cleavage factors; GreB; anti-GreA factor; Gfh1
The Escherichia coli gene encoding the transcription cleavage factor GreB and the Thermus thermophilus gene encoding the anti-GreA transcription factor Gfh1 were cloned and expressed and the purified proteins were crystallized by the sitting-drop vapor-diffusion technique. The GreB and Gfh1 crystals, which were improved by macroseeding, belong to space group P41212 (or P43212), with unit-cell parameters a = b = 148, c = 115.2 Å and a = b = 59.3, c = 218.9 Ǻ, respectively. Complete diffraction data sets were collected for the GreB and Gfh1 crystals to 2.6 and 2.8 Å resolution, respectively. Crystals of the selenomethionine proteins were obtained by microseeding using the native protein crystals and diffract as well as the native ones. The structure determination of these proteins is now in progress.
Several crystals of the Grb2-like C-terminal SH3 domain in complex with a motif peptide from the SLP-76 protein were obtained and characterized.
The Grb2-like adaptor protein GADS is composed of an N-terminal SH3 domain, an SH2 domain, a proline-rich region and a C-terminal SH3 domain. GADS interacts through its C-terminal SH3 domain with the adaptor protein SLP-76, thus recruiting this protein and other associated molecules to the linker for activation of T-cell (LAT) protein. The DNA encoding the C-terminal SH3 domain of GADS (GADS-cSH3) was assembled synthetically using a recursive PCR technique and the protein was overexpressed in Escherichia coli, refolded and purified. Several crystals of this domain in complex with the SLP-76 peptide were obtained and characterized.
GADS; SLP-76; SH3 domain; SH3 dimerization; signal transduction
The endo-1,3-β-glucanase from alkaliphilic Nocardiopsis sp. strain F96 has been crystallized by the hanging-drop vapour-diffusion method. Diffraction data have been collected to 1.3 Å resolution.
Endo-1,3-β-glucanase, an enzyme that hydrolyzes the 1,3-β-glycosyl linkages of β-glucan, belongs to the family 16 glycosyl hydrolases, which are widely distributed among bacteria, fungi and higher plants. Crystals of a family 16 endo-1,3-β-glucanase from the alkaliphilic Nocardiopsis sp. strain F96 were obtained by the hanging-drop vapour-diffusion method. The crystals belonged to space group P21, with unit-cell parameters a = 34.59, b = 71.84, c = 39.67 Å, β = 90.21°, and contained one molecule per asymmetric unit. The Matthews coefficient (V
M) and solvent content were 1.8 Å3 Da−1 and 31.8%, respectively. Diffraction data were collected to a resolution of 1.3 Å and gave a data set with an overall R
merge of 6.4% and a completeness of 99.3%.
family 16 endo-1,3-β-glucanase
A putative ribosomal RNA-processing factor consisting of two KH domains from Pyrococcus horikoshii OT3 (PH1566; 25 kDa) was crystallized by the sitting-drop vapour-diffusion method using PEG 3000 as the precipitant. The space group of the crystals was determined as primitive orthorhombic P212121, with unit-cell parameters a = 45.9, b = 47.4, c = 95.7 Å.
A putative ribosomal RNA-processing factor consisting of two KH domains from Pyrococcus horikoshii OT3 (PH1566; 25 kDa) was crystallized by the sitting-drop vapour-diffusion method using PEG 3000 as the precipitant. The crystals diffracted X-rays to beyond 2.0 Å resolution using a synchrotron-radiation source. The space group of the crystals was determined as primitive orthorhombic P212121, with unit-cell parameters a = 45.9, b = 47.4, c = 95.7 Å. The crystals contain one molecule in the asymmetric unit (V
M = 2.5 Å3 Da−1) and have a solvent content of 50%.
PH1566; RNA-processing factors
The purification, detergent-exchange protocol and crystallization conditions that led to the discovery of HPBP are reported. HPBP is a new human apoprotein that is absent from the genomic database and is the first phosphate transporter characterized in human plasma.
Human phosphate-binding protein (HPBP) was serendipitously discovered by crystallization and X-ray crystallography. HPBP belongs to a eukaryotic protein family named DING that is systematically absent from the genomic database. This apoprotein of 38 kDa copurifies with the HDL-associated apoprotein paraoxonase (PON1) and binds inorganic phosphate. HPBP is the first identified transporter capable of binding phosphate ions in human plasma. Thus, it may be regarded as a predictor of phosphate-related diseases such as atherosclerosis. In addition, HPBP may be a potential therapeutic protein for the treatment of such diseases. Here, the purification, detergent-exchange protocol and crystallization conditions that led to the discovery of HPBP are reported.
ABC transporters; missing gene; apoproteins; atherosclerosis; paraoxonase
MosA from S. meliloti L5-30 has been crystallized in solution with pyruvate and the 2.3 Å resolution structure has been solved by molecular replacement using E. coli dihydrodipicolinate synthase as the model.
The structure of MosA, a dihydrodipicolinate synthase and reported methyltransferase from Sinorhizobium meliloti, has been solved using molecular replacement with Escherichia coli dihydrodipicolinate synthase as the model. A crystal grown in the presence of pyruvate diffracted X-rays to 2.3 Å resolution using synchrotron radiation and belonged to the orthorhombic space group C2221, with unit-cell parameters a = 69.14, b = 138.87, c = 124.13 Å.
dihydrodipicolinate synthase; rhizopine; nitrogen fixation; aldolase
The tRNase domain of colicin D, which is specific to tRNAArgs, has been crystallized. A diffraction data set has been collected to a resolution of 1.05 Å.
The tRNase domain of colicin D, which cleaves only tRNAArgs at the 3′ side of their anticodon loops, has been expressed in Escherichia coli with its inhibitor protein and purified to a form free from the inhibitor using a low-pH buffer. Crystals were obtained by the hanging-drop vapour-diffusion method at 278 K from a buffer containing 100 mM Tris–HCl pH 8.5, 22% PEG MME 2000 and 1 mM nickel(II) chloride. Diffraction data to 1.05 Å resolution were collected at BL41XU, SPring-8. The crystals belong to space group P212121, with unit-cell parameters a = 34.7, b = 65.5, c = 96.5 Å.
arginine tRNAs; atomic resolution; colicin D; tRNases
The arginine repressor of the hyperthermophile T. neapolitana was crystallized with and without its corepressor arginine. Both crystals diffracted to high resolution and belong to the orthorhombic space group P212121, with similar unit-cell parameters.
The arginine repressor of Thermotoga neapolitana (ArgRTnp) is a member of the family of multifunctional bacterial arginine repressors involved in the regulation of arginine metabolism. This hyperthermophilic repressor shows unique DNA-binding features that distinguish it from its homologues. ArgRTnp exists as a homotrimeric protein that assembles into hexamers at higher protein concentrations and/or in the presence of arginine. ArgRTnp was crystallized with and without its corepressor arginine using the hanging-drop vapour-diffusion method. Crystals of the aporepressor diffracted to a resolution of 2.1 Å and belong to the orthorhombic P212121 space group, with unit-cell parameters a = 117.73, b = 134.15, c = 139.31 Å. Crystals of the repressor in the presence of its corepressor arginine diffracted to a resolution of 2.4 Å and belong to the same space group, with similar unit-cell parameters.
arginine repressor; regulation of transcription; hyperthermophiles
X-ray crystallographic characterization of rhesus macaque MHC Mamu-A*02 complexed with an immunodominant SIV-Gag nonapeptide.
Simian immunodeficiency virus (SIV) in the rhesus macaque is regarded as a classic animal model, playing a crucial role in HIV vaccine strategies and therapeutics by characterizing various cytotoxic T-lymphocyte (CTL) responses in macaque monkeys. However, the availability of well documented structural reports focusing on rhesus macaque major histocompatibility complex class I (MHC I) molecules remains extremely limited. Here, a complex of the rhesus macaque MHC I molecule (Mamu-A*02) with human β2m and an immunodominant SIV-Gag nonapeptide, GESNLKSLY (GY9), has been crystallized. The crystal diffracts X-rays to 2.7 Å resolution and belongs to space group C2, with unit-cell parameters a = 124.11, b = 110.45, c = 100.06 Å, and contains two molecules in the asymmetric unit. The availability of the structure, which is being solved by molecular replacement, will provide new insights into rhesus macaque MHC I (Mamu-A*02) presenting pathogenic SIV peptides.
rhesus macaque; simian immunodeficiency virus (SIV); major histocompatibility complex class I (MHC I); Mamu-A*02
The extracellular domain of the 4-1BB ligand fused with glutathione-S-transferase was expressed in Escherichia coli (Origami) and purified by using affinity and ion-exchange column chromatographic methods. Crystals of the 4-1BB ligand were obtained at 290 K by the hanging-drop vapour-diffusion method.
The 4-1BB ligand, a member of the tumour necrosis factor (TNF) family, is an important co-stimulatory molecule that plays a key role in the clonal expansion and survival of CD8+ T cells. Signalling through binding of the 4-1BB ligand and 4-1BB has been reported to enhance CD8+ T-cell expansion and protect activated CD8+ T cells from death. The 4-1BB ligand is an integral protein expressed on activated antigen-presenting cells. The extracellular domain of the 4-1BB ligand fused with glutathione-S-transferase was expressed in Escherichia coli (Origami) and purified by using affinity and ion-exchange column chromatographic methods. Crystals of the 4-1BB ligand were obtained at 290 K by the hanging-drop vapour-diffusion method. X-ray diffraction data were collected from these crystals to 2.8 Å resolution and the crystals belong to space group C2, with unit-cell parameters a = 114.6, b = 73.8, c = 118.50 Å, β = 115.5°.
TNF; 4-1BB ligand; T cells; co-stimulation
Human RuvB-like protein RuvBL1 plays important roles in essential signaling pathways like c-Myc and Wnt, in transcription, and in DNA repair and apoptosis. Crystals of both native and a Se-Met derivative were obtained and characterized. SAD data leading to the structure solution at 2.2 Å were measured from the Se-Met crystals.
RuvBL1, an evolutionary highly conserved protein related to the AAA+ family of ATPases, has been crystallized using the hanging-drop vapour-diffusion method at 293 K. The crystals are hexagonal and belong to space group P6, with unit-cell parameters a = b = 207.1, c = 60.7 Å and three molecules in the asymmetric unit.
The expression, purification, and crystallization in space group P212121 of the complex HasA-HasR from S. marcescens are reported. Diffraction data have been collected and processed to 6.8 Å.
Serratia marcescens is able to acquire iron using its haem-acquisition system (‘has’), which contains an outer membrane receptor HasR and a soluble haemophore HasA. After secretion, HasA binds free haem in the extracellular medium or extracts it from haemoproteins and delivers it to the receptor. Here, the crystallization of a HasA–HasR complex is reported. HasA and HasR have been overexpressed in Escherichia coli and the complex formed and crystallized. Small platelets and bunches of needles of dimensions 0.01 × 0.1 × 1 mm were obtained. A native data set has been collected to 6.8 Å.
HasR; HasA; TonB; haem acquisition; outer-membrane receptor
Dihydropyrimidinase from the slime mould D. discoideum was crystallized. A single crystal was shown to belong to space group I222 and diffracted anisotropically to better than 1.8 Å.
Dihydropyrimidinase (EC 220.127.116.11) is the second enzyme in the reductive pyrimidine-degradation pathway and catalyses the hydrolysis of 5,6-dihydrouracil and 5,6-dihydrothymine to the corresponding N-carbamylated β-amino acids. The recombinant enzyme from the slime mould Dictyostelium discoideum was overexpressed, purified and crystallized by the vapour-diffusion method. One crystal diffracted to better than 1.8 Å resolution on a synchrotron source and was shown to belong to space group I222, with unit-cell parameters a = 84.6, b = 89.6, c = 134.9 Å and one molecule in the asymmetric unit.
dihydropyrimidinase; pyrimidine catabolism; Dictyostelium discoideum
Monoclinic (P21) crystals of a His-tagged form of V. salmonicida catalase without cofactor diffract X-rays to 1.96 Å.
Catalase (EC 18.104.22.168) catalyses the breakdown of hydrogen peroxide to water and molecular oxygen. Recombinant Vibrio salmonicida catalase (VSC) possesses typical cold-adapted features, with higher catalytic efficiency, lower thermal stability and a lower temperature optimum than its mesophilic counterpart from Proteus mirabilis. Crystals of VSC were produced by the hanging-drop vapour-diffusion method using ammonium sulfate as precipitant. The crystals belong to the monoclinic space group P21, with unit-cell parameters a = 98.15, b = 217.76, c = 99.28 Å, β = 110.48°. Data were collected to 1.96 Å and a molecular-replacement solution was found with eight molecules in the asymmetric unit.
Vibrio salmonicida catalase; psychrophiles; cold adaptation
Clitocypin is a cysteine protease inhibitor from the mushroom Clitocybe nebularis. The protein has been purified from natural sources and crystallized in a variety of non-isomorphous forms belonging to monoclinic and triclinic space groups.
Clitocypin is a cysteine protease inhibitor from the mushroom Clitocybe nebularis. The protein has been purified from natural sources and crystallized in a variety of non-isomorphous forms belonging to monoclinic and triclinic space groups. A diffraction data set to 1.55 Å resolution was obtained from a crystal belonging to space group P2, with unit-cell parameters a = 38.326, b = 33.597, c = 55.568 Å, β = 104°. An inability to achieve isomorphism forced the use of MAD and SAD phasing methods. Phasing is in progress.
cysteine protease inhibitors; clitocypin
The P. rubrum sucrose isomerase SmuA, a key enzyme in the industrial production of isomaltulose, was crystallized and diffraction data were collected to 1.95 Å resolution.
Palatinose (isomaltulose, α-d-glucosylpyranosyl-1,6-d-fructofuranose), a nutritional and acariogenic reducing sugar, is industrially obtained from sucrose by using immobilized cells of Protaminobacter rubrum that produce the sucrose isomerase SmuA. The isomerization of sucrose catalyzed by this enzyme also results in the formation of trehalulose (α-d-glucosylpyranosyl-1,1-d-fructofuranose) in smaller amounts and glucose, fructose and eventually isomaltose as by-products, which lower the yield of the reaction and complicate the recovery of palatinose. The determination of the three-dimensional structure of SmuA will provide a basis for rational protein-engineering studies in order to optimize the industrial production of palatinose. A recombinant form of the 67.3 kDa SmuA enzyme has been crystallized in the native state by the vapour-diffusion method. Crystals belong to the orthorhombic space group P212121, with unit-cell parameters a = 61.6, b = 81.4, c = 135.6 Å, and diffract to 1.95 Å resolution on a synchrotron-radiation source.
isomaltulose; industrial strain; sucrose isomerase; glycoside hydrolase family 13
X-ray diffraction data were collected to 1.9 Å from crystals of HLA-G. Cobalt ions were found to be essential for the production of diffracting crystals.
HLA-G is a nonclassical class I major histocompatibility complex (MHC) molecule that is primarily expressed at the foetal–maternal interface. Although the role of HLA-G has not been fully elucidated, current evidence suggests it protects the foetus from the maternal immune response. In this report, HLA-G (44 kDa) is characterized by expression in Escherichia coli. The inclusion bodies were refolded in complex with a peptide derived from histone H2A (RIIPRHLQL), purified and subsequently crystallized. Correct refolding was determined using two conformation-dependent antibodies. Cobalt ions were shown to be an essential ingredient for obtaining diffraction-quality crystals. The crystals, which diffracted to 1.9 Å resolution, belonged to space group P3221, with unit-cell parameters a = b = 77.15, c = 151.72 Å.
class I major histocompatibility complexes; HLA-G
Human common-type acylphosphatase was crystallized in space group P212121, with unit-cell parameters a = 42.58, b = 47.23, c = 57.26 Å. A complete diffraction data set was collected to 1.45 Å.
Human acylphosphatase, an 11 kDa enzyme that catalyzes the hydrolysis of carboxyl phosphate bonds, has been studied extensively as a model system for amyloid-fibril formation. However, the structure is still not known of any isoform of human acylphosphatase. Here, the crystallization and preliminary X-ray diffraction data analysis of human common-type acylphosphatase are reported. Crystals of human common-type acylphosphatase have been grown by the sitting-drop vapour-diffusion method at 289 K using polyethylene glycol 4000 as precipitant. Diffraction data were collected to 1.45 Å resolution at 100 K. The crystals belong to space group P212121, with unit-cell parameters a = 42.58, b = 47.23, c = 57.26 Å.
amyloid fibrils; protein folding; acylphosphatases
The olfactomedin (OLF) domain from the sea urchin cell-adhesion protein amassin has been crystallized. A native data set extending to 2.7 Å has been collected using an in-house X-ray source.
A family of animal proteins is emerging which contain a conserved protein motif known as an olfactomedin (OLF) domain. Novel extracellular protein–protein interactions occur through this domain. The OLF-family member amassin, from the sea urchin Strongylocentrotus purpuratus, has previously been identified to mediate a rapid cell-adhesion event resulting in a large aggregation of coelomocytes, the circulating immune cells. In this work, heterologous expression and purification of the OLF domain from amassin was carried out and initial crystallization trials were performed. A native data set has been collected, extending to 2.7 Å under preliminary cryoconditions, using an in-house generator. This work leads the way to the determination of the first structure of an OLF domain.
olfactomedin domain; cell adhesion; disulfides; blood coagulation; extracellular matrix proteins
Success in crystallization of a functionally intact Hsp70 chaperone required genetic engineering to minimize polydispersity and modulate interdomain interactions, as well as high concentrations of the potent structure stabilizer TMAO. These approaches may be generally useful in crystallization of conformationally flexible proteins that exhibit interdomain motions.
Hsp70s are essential chaperones with roles in a variety of cellular processes and representatives in all kingdoms of life. They are comprised of a nucleotide-binding domain (NBD) and a protein substrate-binding domain (SBD). Structures of isolated NBDs and SBDs have been reported but, until recently, a functionally intact Hsp70 containing both the NBD and SBD has resisted structure determination. Here, it is reported that preparation of diffraction-quality crystals of functionally intact bovine Hsc70 required (i) deletion of part of the protein to reduce oligomerization, (ii) point mutations in the interface between the SBD and NBD and (iii) use of high concentrations of the structure-stabilizing agents glycerol and trimethylamine oxide (TMAO). The introduction of point mutations in interdomain interfaces and the use of the potent structure stabilizer TMAO may be generally useful in crystallization of multidomain proteins that exhibit interdomain motions.
Hsp70; Hsc70; chaperones
A histidine acid phosphatase from the CDC Category A pathogen F. tularensis has been crystallized in space group P41212, with unit-cell parameters a = 61.96, c = 210.78 Å. A 1.75 Å resolution data set was collected at Advanced Light Source beamline 4.2.2.
Francisella tularensis is a highly infectious bacterial pathogen that is considered by the Centers for Disease Control and Prevention to be a potential bioterrorism weapon. Here, the crystallization of a 37.2 kDa phosphatase encoded by the genome of F. tularensis subsp. holarctica live vaccine strain is reported. This enzyme shares 41% amino-acid sequence identity with Legionella pneumophila major acid phosphatase and contains the RHGXRXP motif that is characteristic of the histidine acid phosphatase family. Large diffraction-quality crystals were grown in the presence of Tacsimate, HEPES and PEG 3350. The crystals belong to space group P41212, with unit-cell parameters a = 61.96, c = 210.78 Å. The asymmetric unit is predicted to contain one protein molecule, with a solvent content of 53%. A 1.75 Å resolution native data set was recorded at beamline 4.2.2 of the Lawrence Berkeley National Laboratory Advanced Light Source. Molecular-replacement trials using the human prostatic acid phosphatase structure as the search model (28% amino-acid sequence identity) did not produce a satisfactory solution. Therefore, the structure of F. tularensis histidine acid phosphatase will be determined by multiwavelength anomalous dispersion phasing using a selenomethionyl derivative.
Francisella tularensis; acid phosphatases; histidine acid phosphatases; Legionella pneumophila major acid phosphatase; human prostatic acid phosphatase; RHGXRXP motif
The atomic resolution crystal structure of the double mutant (K53,56M) of bovine pancreatic phospholipase A2 is reported.
The structure of the double mutant K53,56M has previously been refined at 1.9 Å resolution using room-temperature data. The present paper reports the crystal structure of the same mutant K53,56M refined against 1.1 Å data collected using synchrotron radiation. A total of 116 main-chain atoms from 29 residues and 44 side chains are modelled in alternate conformations. Most of the interfacial binding residues are found to be disordered and alternate conformations could be recognized. The second calcium ion-binding site residue Glu92 adopts two alternate conformations. The minor and major conformations of Glu92 correspond to the second calcium ion bound and unbound states.