Acta Crystallographica Section F: Structural Biology and Crystallization Communications

Notes for authors 2012

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Crystallization and X-ray diffraction analysis of a novel surface-adhesin protein: protein E from Haemophilus influenzae

Protein E (PE) is a ubiquitous multifunctional surface protein of Haemophilus spp. and other bacterial pathogens of the Pasteurellaceae family. H. influenzae utilizes PE for attachment to respiratory epithelial cells. In addition, PE interacts directly with plasminogen and the extracellular matrix (ECM) components vitronectin and laminin. Vitronectin is a complement regulator that inhibits the formation of the membrane-attack complex (MAC). PE-mediated vitronectin recruitment at the H. influenzae surface thus inhibits MAC and protects against serum bactericidal activity. Laminin is an abundant ECM protein and is present in the basement membrane that helps in adherence of H. influenzae during colonization. Here, the expression, purification and crystallization of and the collection of high-resolution data for this important H. influenzae adhesin are reported. To solve the phase problem for PE, Met residues were introduced and an SeMet variant was expressed and crystallized. Both native and SeMet-containing PE gave plate-like crystals in space group P21, with unit-cell parameters a = 44, b = 57, c = 61 Å, β = 96°. Diffraction data collected from native and SeMet-derivative crystals extended to resolutions of 1.8 and 2.6 Å, respectively.

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Crystallization and preliminary X-ray analysis of the Clostridium botulinum type D nontoxic nonhaemagglutinin

Clostridium botulinum produces botulinum neurotoxin (BoNT) as a large toxin complex assembled with nontoxic nonhaemagglutinin (NTNHA) and/or haemagglutinin components. Complex formation with NTNHA is considered to be critical in eliciting food poisoning because the complex shields the BoNT from the harsh conditions in the digestive tract. In the present study, NTNHA was expressed in Escherichia coli and crystallized. Diffraction data were collected to 3.9 Å resolution. The crystal belonged to the trigonal space group P321 or P3121/P3221, with unit-cell parameters a = b = 147.85, c = 229.74 Å. The structure of NTNHA will provide insight into the assembly mechanism that produces the unique BoNT–NTNHA complex.

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Refolding, purification and crystallization of the FrpB outer membrane iron transporter from Neisseria meningitidis

FrpB is an integral outer membrane protein from the human pathogen Neisseria meningitidis. It is a member of the TonB-dependent transporter family and promotes the uptake of iron across the outer membrane. There is also evidence that FrpB is an antigen and hence a potential component of a vaccine against meningococcal meningitis. FrpB incorporating a polyhistidine tag was overexpressed in Escherichia coli into inclusion bodies. The protein was then solubilized in urea, refolded and purified to homogeneity. Two separate antigenic variants of FrpB were crystallized by sitting-drop vapour diffusion. Crystals of the F5-1 variant diffracted to 2.4 Å resolution and belonged to space group C2, with unit-cell parameters a = 176.5, b = 79.4, c = 75.9 Å, β = 98.3°. Crystal-packing calculations suggested the presence of a monomer in the asymmetric unit. Crystals of the F3-3 variant also diffracted to 2.4 Å resolution and belonged to space group P212121, with unit-cell parameters a = 85.3, b = 104.6, c = 269.1 Å. Preliminary analysis suggested the presence of an FrpB trimer in the asymmetric unit.

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Purification, crystallization and preliminary X-ray diffraction analysis of the Hsp40 protein CPIP1 from Nicotiana tabacum

Chaperones promote many different molecular processes, including the folding, targeting and degradation of proteins. The best-studied chaperone system consists of the Hsp70s and their co-chaperones the Hsp40s. Chaperone function can be hijacked by viruses in plants. Potato virus Y interacts via its coat protein with an Hsp40 from Nicotiana tabacum, referred to as NtCPIP1, in order to regulate replication. To understand the molecular determinants of this mechanism, different variants of NtCPIP1 were expressed, purified and crystallized. While crystals of wild-type NtCPIP1 diffracted to 8.0 Å resolution, the deletion mutant NtCPIP1-Δ(1:127) crystallized in space group P21212 and diffracted to 2.4 Å resolution.

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Expression, purification, crystallization and preliminary X-ray diffraction analysis of a ribokinase from the thermohalophile Halothermothrix orenii

A ribokinase gene (rbk) from the anaerobic halothermophilic bacterium Halothermothrix orenii was cloned and overexpressed in Escherichia coli. The recombinant protein (Ho-Rbk) was purified using immobilized metal-ion affinity chromatography and crystals were obtained using the sitting-drop method. Diffraction data were collected to a resolution of 3.1 Å using synchrotron radiation. The crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 45.6, b = 61.1, c = 220.2, and contained two molecules per asymmetric unit. A molecular-replacement solution has been found and attempts are currently under way to build a model of the ribokinase. Efforts to improve crystal quality so that higher resolution data can be obtained are also being considered.

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Crystallization and preliminary X-ray diffraction analysis of orotate phosphoribosyltransferase from the human malaria parasite Plasmodium falciparum

Orotate phosphoribosyltransferase (OPRT) catalyzes the Mg2+-dependent condensation of orotic acid (OA) with 5-α-d-phosphorylribose 1-diphosphate (PRPP) to yield diphosphate (PPi) and the nucleotide orotidine 5′-monophosphate. OPRT from Plasmodium falciparum produced in Escherichia coli was crystallized by the sitting-drop vapour-diffusion method in complex with OA and PRPP in the presence of Mg2+. The crystal exhibited tetragonal symmetry, belonging to space group P41 or P43, with unit-cell parameters a = b = 49.15, c = 226.94 Å. X-ray diffraction data were collected to 2.5 Å resolution at 100 K using a synchrotron-radiation source.

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Crystallization and preliminary X-ray diffraction analysis of the metalloregulatory protein DtxR from Thermoplasma acidophilum

The diphtheria toxin repressor (DtxR) is a metal-ion-dependent transcriptional regulator which regulates genes encoding proteins involved in metal-ion uptake to maintain metal-ion homeostasis. DtxR from Thermoplasma acidophilum was cloned and overexpressed in Escherichia coli. Crystals of N-terminally His-tagged DtxR were obtained by hanging-drop vapour diffusion and diffracted to 1.8 Å resolution. DtxR was crystallized at 296 K using polyethylene glycol 4000 as a precipitant. The crystals belonged to the orthorhombic space group P21212, with unit-cell parameters a = 61.14, b = 84.61, c = 46.91 Å, α = β = γ = 90°. The asymmetric unit contained approximately one monomer of DtxR, giving a crystal volume per mass (VM) of 2.22 Å3 Da−1 and a solvent content of 44.6%.

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Crystallization and preliminary crystallographic analysis of LipC12, a true lipase isolated through a metagenomics approach

LipC12, a true lipase from family I.1 of bacterial lipases which was previously isolated through a metagenomics approach, contains 293 amino acids. Among lipases of known three-dimensional structure, it has a sequence identity of 47% to the lipase from Pseudomonas aeruginosa PAO1. Recombinant N-terminally His6-tagged LipC12 protein was expressed in Escherichia coli, purified in a homogenous form and crystallized in several conditions, with the best crystals being obtained using 2.0 M sodium formate and 0.1 M bis-tris propane pH 7.0. X-ray diffraction data were collected to 2.70 Å resolution. The crystals belonged to the tetragonal space group P4122, with unit-cell parameters a = b = 58.62, c = 192.60 Å.

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Purification, crystallization and preliminary X-ray crystallographic studies of the Mycobacterium tuberculosis DNA gyrase CTD

Mycobacterium tuberculosis DNA gyrase, a nanomachine involved in regulation of DNA topology, is the only type II topoisomerase present in this organism and hence is the sole target of fluoroquinolone in the treatment of tuberculosis. The C-terminal domain (CTD) of the DNA gyrase A subunit possesses a unique feature, the ability to wrap DNA in a chiral manner, that plays an essential role during the catalytic cycle. A construct of 36 kDa corresponding to this domain has been overproduced, purified and crystallized. Diffraction data were collected to 1.55 Å resolution. Cleavage of the N-terminal His tag was crucial for obtaining crystals. The crystals belonged to space group P212121, with one molecule in the asymmetric unit and a low solvent content (33%). This is the first report of the crystallization and preliminary X-ray diffraction studies of a DNA gyrase CTD from a species that contains one unique type II topoisomerase.

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Expression, purification, crystallization and preliminary X-ray analysis of Pseudomonas aeruginosa PelD

The production of the PEL polysaccharide in Pseudomonas aeruginosa requires the binding of bis-(3′,5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) to the cytoplasmic GGDEF domain of the inner membrane protein PelD. Here, the overexpression, purification and crystallization of a soluble construct of PelD that encompasses the GGDEF domain and a predicted GAF domain is reported. Diffraction-quality crystals were grown using the hanging-drop vapour-diffusion method. The crystals grew as flat plates, with unit-cell parameters a = 88.3, b = 114.0, c = 61.9 Å, α = β = γ = 90.0°. The PelD crystals exhibited the symmetry of space group P21212 and diffracted to a minimum d-spacing of 2.2 Å. On the basis of the Matthews coefficient (VM = 2.29 Å3 Da−1), it was estimated that two molecules are present in the asymmetric unit.

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Crystallization of oligonucleotides containing A-rich repeats suggests a structural contribution to the autoregulation mechanism of PABP translation

Eukaryotic poly(A)-binding protein (PABP) commonly binds to the 3′-UTR poly(A) tail of every mRNA, but it also binds to the 5′-UTR of PABP mRNA for autoregulation of its expression. In the sequence of the latter binding site, the contiguous A residues are segmented discretely by the insertion of short pyrimidine oligonucleotides as linkers, so that (A)6–8 segments are repeated six times. This differs from the poly(A)-tail sequence, which has a higher binding affinity for PABP. In order to examine whether the A-rich repeats have a functional structure, several RNA/DNA analogues were subjected to crystallization. It was found that some of them could be crystallized. Single crystals thus obtained diffracted to 4.1 Å resolution. The fact that the repeated sequences can be crystallized suggests the possibility that the autoregulatory sequence in PABP mRNA has a specific structure which impedes the binding of PABP. When PABP is excessively produced, it could bind to this sequence by releasing the structure in order to interfere with initiation-complex formation for suppression of PABP translation. Otherwise, PABP at low concentration preferentially binds to the poly(A) tail of PABP mRNA.

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Crystallization and X-ray diffraction analysis of chondroitin lyase from baculovirus: envelope protein ODV-E66

Baculovirus envelope protein ODV-E66 (67–704), in which the N-terminal 66 amino acids are truncated, is a chondroitin lyase. It digests chondroitin and chondroitin 6-sulfate efficiently, but does not digest chondroitin 4-sulfate. This unique characteristic is useful for the preparation of specific chondroitin oligosaccharides and for investigation of the mechanism of baculovirus infection. ODV-E66 (67–704) was crystallized; the crystal diffracted to 1.8 Å resolution and belonged to space group P62 or P64, with unit-cell parameters a = b = 113.5, c = 101.5 Å. One molecule is assumed to be present per asymmetric unit, which gives a Matthews coefficient of 2.54 Å3 Da−1.

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Crystallization and preliminary X-ray crystallographic analysis of dioscorin from Dioscorea japonica

Dioscorin, the major tuber storage protein in yam, has been reported to possess carbonic anhydrase, trypsin inhibitor, angiotensin-converting enzyme (ACE) inhibitor, free-radical scavenger, dehydroascorbate reductase and monodehydroascorbate reductase activities. Recent research has also found that dioscorin can enhance immune modulation via the toll-like receptor 4 (TLR-4) signal transduction pathway in RAW 264.7 cells, murine bone-marrow cells and human monocytes ex vivo. Resolving the structure of dioscorin would help in better understanding its activities and would provide clues to understanding the mechanism of its multiple functions. The full-length protein (residues 1–246) with an additional His6 tag at the N-terminus was expressed in Escherichia coli Rosetta (DE3) cells. After His-tag cleavage and purification, the protein was crystallized by the sitting-drop vapour-diffusion method at 278 K. An X-ray diffraction data set was collected to a resolution of 2.11 Å using a synchrotron X-ray source. The crystal belonged to space group C2221, with unit-cell parameters a = 83.5, b = 156.8, c = 83.6 Å, and was estimated to contain two protein molecules per asymmetric unit.

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Purification, crystallization and preliminary X-ray characterization of Bacillus cereus arylamine N-acetyltransferase 3 [(BACCR)NAT3]

Arylamine N-acetyltransferases (NATs) are xenobiotic metabolizing enzymes (XMEs) that catalyze the acetylation of arylamines. All functional NATs described to date possess a strictly conserved Cys-His-Asp catalytic triad. Here, the purification, crystallization and preliminary X-ray characterization of Bacillus cereus arylamine N-acetyltransferase 3 [(BACCR)NAT3], a putative NAT isoenzyme that possesses a unique catalytic triad containing a glutamate residue, is reported. The crystal diffracted to 2.42 Å resolution and belonged to the monoclinic space group C121, with unit-cell parameters a = 90.44, b = 44.52, c = 132.98 Å, β = 103.8°.

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Crystallization and preliminary X-ray analysis of S-ribosylhomocysteinase from Streptococcus mutans

S-Ribosylhomocysteinase (LuxS) encoded by the luxS gene from Streptococcus mutans plays a crucial role in the quorum-sensing system. LuxS was solubly expressed in Escherichia coli with high yield. The purity of the purified target protein, which was identified by SDS–PAGE and MALDI–TOF MS analysis, was >95%. The protein was crystallized using the hanging-drop vapour-diffusion method with PEG 3350 as the primary precipitant. X-ray diffraction data were collected at Beijing Synchrotron Radiation Facility (BSRF). Diffraction by the crystal extended to 2.4 Å resolution and the crystal belonged to space group C2221, with unit-cell parameters a = 55.3, b = 148.7, c = 82.8 Å.

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Crystallization and preliminary X-ray diffraction analysis of Bacillus subtilis YwfE, an l-amino-acid ligase

Bacillus subtilis YwfE, an l-amino-acid ligase, catalyzes the formation of an α-dipeptide from l-amino acids in an ATP-dependent manner. In order to elucidate the substrate-recognition mode and the reaction mechanism of this ligase, native and selenomethionine-derivatized (SeMet) crystals of YwfE in the presence of ADP, MgCl2 and the dipeptide l-Ala-l-Gln were obtained using the hanging-drop vapour-diffusion method. These crystals diffracted to 1.9 and 2.8 Å resolution, respectively. Preliminary SAD phase calculations using the data set from the SeMet crystal suggested that the crystal belonged to the hexagonal space group P6522, with unit-cell parameters a = b = 90.85, c = 250.31 Å, and contained one molecule in the asymmetric unit with a solvent content of 57.3%.

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Cloning, expression, crystallization and preliminary X-ray crystallographic analysis of aspartyl aminopeptidase from the apeB gene of Pseudomonas aeruginosa

Aminopeptidases (APs) are a group of exopeptidases that catalyze the removal of amino acids from the N-termini of proteins and peptides. The APs are ubiquitous in nature and are of critical biological and medical importance because of their key role in protein degradation. Pseudomonas aeruginosa aspartyl aminopeptidase (PaAAP), which is encoded by the apeB gene, was expressed in Escherichia coli, purified and crystallized using the microbatch method. A preliminary structural study has been performed using the X-ray crystallographic method. The PaAAP crystal diffracted to 2.0 Å resolution and belonged to the rhombohedral space group H3, with unit-cell parameters a = b = 133.6, c = 321.2. The unit-cell volume of the crystal is compatible with the presence of four monomers in the asymmetric unit, with a corresponding Matthews coefficient VM of 2.95 Å3 Da−1 and a solvent content of 58.3%.

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Crystallization and preliminary X-ray diffraction analysis of an l-amino-acid oxidase from Bothrops jararacussu venom

Snake-venom l-amino-acid oxidases (SV-LAAOs) trigger a wide range of local and systematic effects, including inhibition of platelet aggregation, cytotoxicity, haemolysis, apoptosis and haemorrhage. These effects mainly arise from the uncontrolled release of the hydrogen peroxide that is produced by the redox reaction involving l-amino acids catalyzed by these flavoenzymes. Taking their clinical relevance into account, few SV-LAAOs have been structurally characterized and the structural determinants responsible for their broad direct and indirect pharmacological activities remain unclear. In this work, an LAAO from Bothrops jararacussu venom (BJu-LAAO) was purified and crystallized. The BJu-LAAO crystals belonged to space group P21, with unit-cell parameters a = 66.38, b = 72.19, c = 101.53 Å, β = 90.9°. The asymmetric unit contained two molecules and the structure was determined and partially refined at 3.0 Å resolution.

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Crystallization and preliminary X-ray diffraction analysis of human dipeptidyl peptidase 10 (DPPY), a component of voltage-gated potassium channels

Dipeptidyl peptidase 10 (DPP10, DPPY) is an inactive peptidase associated with voltage-gated potassium channels, acting as a modulator of their electrophysiological properties, cell-surface expression and subcellular localization. Because potassium channels are important disease targets, biochemical and structural characterization of their interaction partners was sought. DPP10 was cloned and expressed using an insect-cell system and the protein was purified via His-tag affinity and size-exclusion chromatography. Crystals obtained by the sitting-drop method were orthorhombic, belonging to space group P212121 with unit-cell parameters a = 80.91, b = 143.73, c = 176.25 Å. A single solution with two molecules in the asymmetric unit was found using the structure of DPP6 (also called DPPX; PDB entry 1xfd) as the search model in a molecular replacement protocol.

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