Serine/threonine protein kinase SnRK2.6/OST1 (OPEN STOMATA 1) from A. thaliana has been crystallized by the hanging-drop vapour-diffusion method and a native data set has been collected to 2.8 Å resolution.
The SnRK2.6 (SNF1-related kinase 2.6) gene from Arabidopsis thaliana encodes the serine/threonine protein kinase SnRK2.6/OST1 (OPEN STOMATA 1). It plays a central role in the drought-tolerance mechanism. OST1 is in fact the main positive effector in the hydric stress response. The SnRK2.6 gene was cloned into the pGEX4T1 plasmid, mutated and expressed in Escherichia coli, allowing purification to homogeneity in two chromatographic steps. Various OST1 mutants yielded crystals using vapour-diffusion techniques, but only one mutant showed a good diffraction pattern. Its crystals diffracted to 2.8 Å resolution and belonged to space group P2221, with unit-cell parameters a = 77.7, b = 99.4, c = 108.4 Å. A promising molecular-replacement solution was found using the structure of the kinase domain of the yeast AMP-activated protein kinase SNF1 (PDB entry 3hyh) as the search model.
SnRK2.6/OST1; serine/threonine protein kinases; Arabidopsis thaliana
TEM-72 is a quadruple mutant of TEM-1 and shows extended-spectrum β-lactamase properties. The present structure shows the presence of a citrate anion bound to the TEM-72 active site and supports the use of polycarboxylates as a scaffold for the design of broad-spectrum inhibitors of serine β-lactamases.
TEM-72, a class A β-lactamase identified in isolates of Enterobacteriaceae, is a quadruple mutant of TEM-1 (Q39K, M182T, G238S and E240K) and shows extended-spectrum β-lactamase (ESBL) properties arising from the G238S and E240K substitutions. Although many structures of TEM variants have been published, they do not include an enzyme with the simultaneous presence of both of the ESBL-conferring G238S and E240K substitutions. Furthermore, the structure shows the presence of a citrate anion bound to the TEM-72 active site, where it interacts with all of the conserved residues of class A β-lactamases. The present structure supports the use of polycarboxylates as a scaffold for the design of broad-spectrum inhibitors of serine β-lactamases.
class A β-lactamases; extended-spectrum β-lactamases; inhibitors; citrate; polycarboxylate
Crystals of SimR, a TetR-like efflux pump repressor from S. antibioticus, were obtained and X-ray data were recorded to a resolution of 2.3 Å.
Crystals of SimR were grown by vapour diffusion. The protein crystallized with trigonal symmetry and X-ray data were recorded to a resolution of 2.3 Å from a single crystal at the synchrotron. SimR belongs to the TetR family of bacterial transcriptional regulators. In the absence of the antibiotic simocyclinone, SimR represses the transcription of a divergently transcribed gene encoding the simocyclinone efflux pump SimX in Streptomyces antibioticus by binding to operators in the simR–simX intergenic region. Simocyclinone binding causes SimR to dissociate from its operators, leading to expression of the SimX efflux pump. Thus, SimR represents an intimate link between the biosynthesis of simocyclinone and its export, which may also provide the mechanism of self-resistance to the antibiotic in the producer strain.
TetR-like repressors; simocyclinone; efflux pumps; antibiotic resistance
The crystallization of an ASCH domain-containing protein from Z. mobilis ZM4 and the collection of diffraction data to 2.1 Å resolution are reported.
The human activating signal cointegrator 1 (ASC-1) homology (ASCH) domain is frequently observed in many organisms, although its function has not yet been clearly defined. In Zymomonas mobilis ZM4, the ZMO0922 gene encodes a polypeptide that includes an ASCH domain (zmASCH). To provide a better structural background for the probable role of ASCH domain-containing proteins, the ZMO0922 gene was cloned and expressed. The purified protein was crystallized from 30%(w/v) polyethylene glycol 400, 0.1 M cacodylic acid pH 6.5 and 0.2 M lithium sulfate. Diffraction data were collected to 2.1 Å resolution using synchrotron radiation. The crystal belonged to the primitive trigonal space group P3121 or P3221, with unit-cell parameters a = b = 51.67, c = 207.30 Å, α = β = 90, γ = 120°. Assuming the presence of one molecule in the asymmetric unit gave a Matthews coefficient of 4.69 Å3 Da−1, corresponding to a solvent content of 73.7%.
ASCH domains; Zymomonas mobilis ZM4
Biliverdin reductase (BVR) from Synechocystis sp. PCC6803 and its selenomethionine derivative were overexpressed and purified. X-ray diffraction data from an SeMet BVR microcrystal were collected to 3.0 Å resolution on microfocus beamline BL32XU at SPring-8.
Biliverdin reductase (BVR) catalyzes the conversion of biliverdin IX α to bilirubin IX α with concomitant oxidation of an NADH or NADPH cofactor. This enzyme also binds DNA and enhances the transcription of specific genes. Recombinant cyanobacterial BVR was overexpressed in Escherichia coli, purified and crystallized. A native data set was collected to 2.34 Å resolution on beamline BL38B1 at SPring-8. An SeMet data set was collected from a microcrystal (300 × 10 × 10 µm) on the RIKEN targeted protein beamline BL32XU and diffraction spots were obtained to 3.0 Å resolution. The native BVR crystal belonged to space group P212121, with unit-cell parameters a = 58.8, b = 88.4, c = 132.6 Å. Assuming that two molecules are present in the asymmetric unit, V
M (the Matthews coefficient) was calculated to be 2.37 Å3 Da−1 and the solvent content was estimated to be 48.1%. The structure of cyanobacterial BVR may provide insights into the mechanisms of its enzymatic and physiological functions.
bilirubin; biliverdin; microcrystals; microfocus beamline
Preliminary crystallographic analysis of the CBS domain protein MJ1004 from Methanocaldococcus jannaschii.
The purification and preliminary crystallographic analysis of the archaeal CBS-domain protein MJ1004 from Methanocaldococcus jannaschii are described. The native protein was overexpressed, purified and crystallized in the monoclinic space group P21, with unit-cell parameters a = 54.4, b = 53.8, c = 82.6 Å, β = 106.1°. The crystals diffracted X-rays to 2.7 Å resolution using synchrotron radiation. Matthews-volume calculations suggested the presence of two molecules in the asymmetric unit that are likely to correspond to a dimeric species, which is also observed in solution.
MJ1004; CBS domains; Bateman domains; Methanocaldococcus jannaschii
The preparative purification and crystallization of Flt3 receptor–ligand complexes are described.
The extracellular complex between the haematopoietic receptor Flt3 and its cytokine ligand (FL) is the cornerstone of signalling cascades that are central to early haematopoiesis and the immune system. Here, efficient protocols for the production of two ectodomain variants of human Flt3 receptor, Flt3D1–D5 and Flt3D1–D4, for structural studies are reported based on tetracycline-inducible stable cell lines in HEK293S cells deficient in N-acetylglycosaminyltransferase I (GnTI−/−) that can secrete the target proteins with limited and homogeneous N-linked glycosylation to milligram amounts. The ensuing preparative purification of Flt3 receptor–ligand complexes yielded monodisperse complex preparations that were amenable to crystallization. Crystals of the Flt3D1–D4–FL and Flt3D1–D5–FL complexes diffracted to 4.3 and 7.8 Å resolution, respectively, and exhibited variable diffraction quality even within the same crystal. The resulting data led to the successful structure determination of Flt3D1–D4–FL via a combination of molecular-replacement and density-modification protocols exploiting the noncrystallographic symmetry and high solvent content of the crystals.
Fms-like tyrosine kinase 3 receptor; Flt3; Flt3 ligand; ectodomains
The first high-angle neutron fibre diffraction results from an amyloid system are described. These were obtained using H2O/D2O isototopic replacement, and emphasise the ability of neutron fibre diffraction, used in conjunction with deuterium labelling approaches, to study novel aspects of amyloid conformation and hydration.
The first neutron fibre diffraction studies of an amyloid system are presented. The techniques used to prepare the large samples needed are described, as well as the procedures used to isotopically replace H2O in the sample by D2O. The results demonstrate the feasibility of this type of approach for the pursuit of novel structural analyses that will strongly complement X-ray fibre diffraction studies and probe aspects of amyloid structure that to date have remained obscure. The approach is demonstrated using an amyloid form of the peptide NSGAITIG, but is equally applicable for the study of other systems such as Alzheimer’s Aβ peptide.
amyloid; neutron fibre diffraction; H2O/D2O isotopic replacement
The Src-homology 2 (SH2) domain of Csk-family protein tyrosine kinases acts as a conformational switch to regulate their catalytic activity, which in turn promotes the inhibition of their proto-oncogenic targets, the Src-family kinases. Here, the expression, purification, small-angle X-ray scattering and preliminary diffraction analysis of the SH2 domain of the Csk-homologous kinase is reported.
The C-terminal Src kinase (Csk) and Csk-homologous kinase (CHK) are endogenous inhibitors of the proto-oncogenic Src family of protein tyrosine kinases (SFKs). Phosphotyrosyl peptide binding to their Src-homology 2 (SH2) domains activates Csk and CHK, enhancing their ability to suppress SFK signalling; however, the detailed mechanistic basis of this activation event is unclear. The CHK SH2 was expressed in Escherichia coli and the purified protein was characterized as monomeric by synchrotron small-angle X-ray scattering in-line with size-exclusion chromatography. The CHK SH2 crystallized in 0.2 M sodium bromide, 0.1 M bis-Tris propane pH 6.5 and 20% polyethylene glycol 3350 and the best crystals diffracted to ∼1.6 Å resolution. The crystals belonged to space group P2, with unit-cell parameters a = 25.8, b = 34.6, c = 63.2 Å, β = 99.4°.
Csk-homologous kinase; Src-homology 2 domains; enzyme inhibition; Src-family protein tyrosine kinases; cancer; small-angle X-ray scattering
Crystals of a chitinase from C. vernus were monoclinic, belonging to space group C2 with unit-cell parameters a = 172.3, b = 37.1, c = 126.4 Å, β = 127°, and diffracted to 2.1 Å resolution.
A chitinase has been isolated and purified from Crocus vernus corms. N-terminal amino-acid sequence analysis of the approximately 30 kDa protein showed 33% identity to narbonin, a seed protein from Vicia narbonensis L. The C. vernus chitinase was crystallized by the hanging-drop vapour-diffusion method using PEG 8000 as the main precipitant. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 172.3, b = 37.1, c = 126.4 Å, β = 127° and two molecules per asymmetric unit. Diffraction data were collected to a resolution of 2.1 Å.
chitinases; Crocus vernus
This work describes the purification and preliminary crystallographic analysis of the CBS-pair regulatory domain of the human ancient domain protein 4 (ACDP4), also known as CNNM4.
This work describes the purification and preliminary crystallographic analysis of the CBS-pair regulatory domain of the human ancient domain protein 4 (ACDP4), also known as CNNM4. ACDP proteins represent the least-studied members of the eight different types of magnesium transporters that have been identified in mammals to date. In humans the ACDP family includes four members: CNNM1–4. CNNM1 acts as a cytosolic copper chaperone and has been associated with urofacial syndrome, whereas CNNM2 and CNNM4 have been identified as magnesium transporters. Interestingly, mutations in the CNNM4 gene have clinical consequences that are limited to retinal function and biomineralization and are considered to be the cause of Jalili syndrome, which consists of autosomal recessive cone-rod dystrophy and amelogenesis imperfecta. The truncated protein was overexpressed, purified and crystallized in the orthorhombic space group C222. The crystals diffracted X-rays to 3.6 Å resolution using synchrotron radiation. Matthews volume calculations suggested the presence of two molecules in the asymmetric unit, which were likely to correspond to a CBS module of the CBS pair of CNNM4.
Mg2+ transporters; CBS domains; Jalili syndrome; biomineralization; sensory transduction; vision; cyclin M4; ion transport; magnesium; ancient conserved domain-containing protein 4
A lactonase from the hyperthermophilic archaeon S. islandicus has been crystallized. In combination with biochemical and bioengineering studies, it is expected that the structure of this protein will provide insight into the natural function of the phosphotriesterase-like lactonase family.
Phosphotriesterase-like lactonases (PLLs) constitute an interesting family of enzymes that are of paramount interest in biotechnology with respect to their catalytic functions. As natural lactonases, they may act against pathogens such as Pseudomonas aeruginosa by shutting down their quorum-sensing system (quorum quenching) and thus decreasing pathogen virulence. Owing to their promiscuous phosphotriesterase activity, which can inactivate toxic organophosphorus compounds such as pesticides and nerve agents, they are equally appealing as potent bioscavengers. A new representative of the PLL family has been identified (SisPox) and its gene was cloned from the hyperthermophilic archeon Sulfolobus islandicus. Owing to its hyperthermostable architecture, SisPox appears to be a good candidate for engineering studies. Here, production, purification, crystallization conditions and data collection to 2.34 Å resolution are reported for this lactonase from the hyperthermophilic S. islandicus.
lactonases; phosphotriesterases; phosphotriesterase-like lactonases; quorum sensing; quorum quenching; bioscavengers; hyperthermophiles
This work demonstrates how the digestion of a surface-exposed loop of B. fragilis type III glutamine synthetase results in crystal-packing rearrangements and explains how these changes facilitated the first structure solution.
This work details the intentional modifications that led to the first structure of a type III glutamine synthetase enzyme (GSIII). This approach followed the serendipitous discovery of digestion caused by an extracellular protease from a contaminating bacterium, Pseudomonas fluorescens. The protease only cleaves the GSIII protein at a single site, leaving the oligomer intact but allowing the protein to crystallize in a different space group. This transition from space group P1 to space group C2221 is accompanied by improved growth characteristics, more reproducible diffraction and enhanced mechanical stability. The crystallographic analyses presented here provide the structural basis of the altered molecular packing in the full-length and digested crystal forms and suggest modifications for future structural studies.
glutamine synthetase III; Bacteroides fragilis
The crystallization of the C. perfringens delta-toxin is reported.
Clostridium perfringens is a Gram-positive anaerobic bacterium that is responsible for a wide range of diseases in humans and both wild and domesticated animals, including birds. C. perfringens is notable for its ability to produce a plethora of toxins, e.g. phospholipases C (alpha-toxin), pore-forming toxins (epsilon-toxin, beta-toxin and enterotoxin) and binary toxins (iota-toxin). Based on alpha-, beta-, epsilon- and iota-toxin production, the bacterium is classified into five different toxinotypes (A–E). Delta-toxin, which is a 32.6 kDa protein with 290 amino acids, is one of three haemolysins released by type C and possibly by type B strains of C. perfringens. This toxin is immunogenic and lytic to erythrocytes from the even-toed ungulates sheep, goats and pigs, and is cytotoxic to other cell types such as rabbit macrophages, human monocytes and blood platelets from goats, rabbits, guinea pigs and humans. The recombinant delta-toxin has been cloned, expressed, purified and crystallized in two different crystal forms by the hanging-drop vapour-diffusion method. Of these two different crystal forms, only the form II crystal diffracted to atomic resolution (d
min = 2.4 Å), while the form I crystal diffracted to only 15 Å resolution. The form II crystals belonged to space group P21212, with one molecule in the crystallographic asymmetric unit and unit-cell parameters a = 49.66, b = 58.48, c = 112.93 Å.
Clostridium perfringens; delta-toxin; haemolysins
Juvenile hormone-binding protein and PBMHP-12, two major 30 kDa proteins, have been isolated and purified from the haemolymph of B. mori. The proteins were crystallized and the crystals diffracted X-rays to 2.9 and 1.3 Å resolution, respectively.
Juvenile hormone-binding protein (JHBP) and the low-molecular-mass lipoprotein PBMHP-12 belong to a group of 30 kDa proteins that comprise the major protein component of the haemolymph specific to the fifth-instar larvae stage of the mulberry silkworm Bombyx mori L. Proteins from this group are often essential for the development of the insect. In a project aimed at crystallographic characterization of B. mori JHBP (BmJHBP), it was copurified together with PBMHP-12. Eventually, the two proteins were isolated and crystallized separately. The BmJHBP crystals were orthorhombic (space group C2221) and the PBMHP-12 crystals were triclinic. The crystals diffracted X-rays to 2.9 Å (BmJHBP) and 1.3 Å (PBMHP-12) resolution.
juvenile hormone-binding protein; PBMHP-12; Bombyx mori; haemolymph
The DNA-binding domain of R. capsulatus MopB has been purified and crystallized for X-ray structure analysis.
The LysR-type regulator MopB represses transcription of several target genes (including the nitrogen-fixation gene anfA) in Rhodobacter capsulatus at high molybdenum concentrations. In this study, the isolated DNA-binding domain of MopB (MopBHTH) was overexpressed in Escherichia coli. Purified MopBHTH bound the anfA promoter as shown by DNA mobility-shift assays, demonstrating the function of the isolated regulator domain. MopBHTH was crystallized using the sitting-drop vapour-diffusion method in the presence of 0.2 M lithium sulfate, 0.1 M phosphate/citrate pH 4.2, 20%(w/v) PEG 1000 at 291 K. The crystal belonged to space group P3121 or P3221, with unit-cell parameters a = b = 61.84, c = 139.64 Å, α = β = 90, γ = 120°, and diffracted to 3.3 Å resolution at a synchrotron source.
MopB; Rhodobacter capsulatus; DNA-binding domain; LysR-type transcription regulators
The regulatory domain of M. tuberculosis aspartokinase, the enzyme which catalyses the first reaction step in the biosynthesis of the amino acids lysine, methionine and threonine, has been cloned, expressed, purified and crystallized. Preliminary X-ray diffraction analysis of several crystals revealed the presence of five distinct crystal forms.
The regulatory domain of Mycobacterium tuberculosis aspartokinase (Mtb-AK, Mtb-Ask, Rv3709c) has been cloned, heterologously expressed in Escherichia coli and purified using standard chromatographic techniques. Screening for initial crystallization conditions using the regulatory domain (AK-β) in the presence of the potential feedback inhibitor threonine identified four conditions which yielded crystals suitable for X-ray diffraction analysis. From these four conditions five different crystal forms of Mtb-AK-β resulted, three of which belonged to the orthorhombic system, one to the tetragonal system and one to the monoclinic system. The highest resolution (1.6 Å) was observed for a crystal form belonging to space group P212121, with unit-cell parameters a = 53.70, b = 63.43, c = 108.85 Å and two molecules per asymmetric unit.
aspartokinase; Rv3709c; Mycobacterium tuberculosis; tuberculosis
The cloning, expression, purification, crystallization and preliminary crystallographic analysis of mannosyl-3-phosphoglycerate phosphatase (MpgP) from T. thermophilus HB27 are reported. The stability of MpgP in solution was studied by size-exclusion chromatography and differential scanning fluorimetry assays.
Mannosylglycerate (MG) is primarily known as an osmolyte and is widely distributed among (hyper)thermophilic marine microorganisms. The synthesis of MG via mannosyl-3-phosphoglycerate synthase (MpgS) and mannosyl-3-phosphoglycerate phosphatase (MpgP), the so-called two-step pathway, is the most prevalent route among these organisms. The phosphorylated intermediate mannosyl-3-phosphoglycerate is synthesized by the first enzyme and is subsequently dephosphorylated by the second. The structure of MpgS from the thermophilic bacterium Thermus thermophilus HB27 has recently been solved and characterized. Here, the cloning, expression, purification, crystallization and preliminary crystallographic analysis of MpgP from T. thermophilus HB27 are reported. Size-exclusion chromatography assays suggested a dimeric assembly in solution for MpgP at pH 6.3 and together with differential scanning fluorimetry data showed that high ionic strength and charge compensation were required to produce a highly pure and soluble protein sample for crystallographic studies. The crystals obtained belonged to the monoclinic space group P21, with unit-cell parameters a = 39.52, b = 70.68, c = 95.42 Å, β = 92.95°. Diffraction data were measured to 1.9 Å resolution. Matthews coefficient calculations suggested the presence of two MpgP monomers in the asymmetric unit and the calculation of a self-rotation Patterson map indicated that the two monomers could be related by a noncrystallographic twofold rotation axis, forming a dimer.
mannosyl-3-phosphoglycerate phosphatase; Thermus thermophilus HB27; mannosylglycerate synthesis
A mutant of the haloalkane dehalogenase DhaA (DhaA31) from R. rhodochrous NCIMB 13064 and its complex with 1,2,3-trichloropropane were crystallized and the crystals diffracted to high resolution.
Haloalkane dehalogenases hydrolyze carbon–halogen bonds in a wide range of halogenated aliphatic compounds. The potential use of haloalkane dehalogenases in bioremediation applications has stimulated intensive investigation of these enzymes and their engineering. The mutant DhaA31 was constructed to degrade the anthropogenic compound 1,2,3-trichloropropane (TCP) using a new strategy. This strategy enhances activity towards TCP by decreasing the accessibility of the active site to water molecules, thereby promoting formation of the activated complex. The structure of DhaA31 will help in understanding the structure–function relationships involved in the improved dehalogenation of TCP. The mutant protein DhaA31 was crystallized by the sitting-drop vapour-diffusion technique and crystals of DhaA31 in complex with TCP were obtained using soaking experiments. Both crystals belonged to the triclinic space group P1. Diffraction data were collected to high resolution: to 1.31 Å for DhaA31 and to 1.26 Å for DhaA31 complexed with TCP.
haloalkane dehalogenases; DhaA; Rhodococcus rhodochrous
Crystallographic data of organic solvent-tolerant lipase from Bacillus sp. strain 42 was collected at 2.0 Å with unit-cell parameters a =117.41, b = 80.85, c = 99.44 Å, β=96.40°. The protein–solvent interactions will be studied since lipase 42 was stable in water-miscible solvent.
An organic solvent-tolerant lipase from Bacillus sp. strain 42 was crystallized using the capillary-tube method. The purpose of studying this enzyme was in order to better understand its folding and to characterize its properties in organic solvents. By initially solving its structure in the native state, further studies on protein–solvent interactions could be performed. X-ray data were collected at 2.0 Å resolution using an in-house diffractometer. The estimated crystal dimensions were 0.09 × 0.19 × 0.08 mm. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 117.41, b = 80.85, c = 99.44 Å, β = 96.40°.
lipases; Bacillus sp. strain 42; organic solvent tolerance
The expression, purification and crystallization of the SARS coronavirus nsp16 RNA-cap AdoMet-dependent (nucleoside-2′O)-methyltransferase in complex with its activating factor nsp10 are reported.
To date, the SARS coronavirus is the only known highly pathogenic human coronavirus. In 2003, it was responsible for a large outbreak associated with a 10% fatality rate. This positive RNA virus encodes a large replicase polyprotein made up of 16 gene products (nsp1–16), amongst which two methyltransferases, nsp14 and nsp16, are involved in viral mRNA cap formation. The crystal structure of nsp16 is unknown. Nsp16 is an RNA-cap AdoMet-dependent (nucleoside-2′-O-)-methyltransferase that is only active in the presence of nsp10. In this paper, the expression, purification and crystallization of nsp10 in complex with nsp16 are reported. The crystals diffracted to a resolution of 1.9 Å resolution and crystal structure determination is in progress.
SARS coronavirus; nsp10; nsp16
4-Coumarate:CoA ligase 2 from A. thaliana has been overexpressed in E. coli, purified and crystallized. Diffraction data were collected to a resolution of 1.6 Å.
4-Coumarate:CoA ligase 2 (4CL2) from Arabidopsis thaliana catalyzes the ATP-dependent formation of the 4-coumaroyl-CoA thioester through the formation of 4-coumarate-AMP. Recombinant 4CL2 protein was expressed in Escherichia coli and crystallized by the sitting-drop vapour-diffusion method. The crystals belonged to space group P21, with unit-cell parameters a = 91.6, b = 55.5, c = 124.4 Å, α = γ = 90.0, β = 111.1°.
CoA ligases; ANL superfamily; phenylpropanoids; lignin biosynthesis
A truncated version of pleckstrin encompassing the amino-terminal PH domain and the central DEP domain has been crystallized and preliminary diffraction analysis has been conducted. The quaternary structure and phospholipid-binding properties of this pleckstrin truncation were analyzed.
Pleckstrin is a major substrate of protein kinase C in platelets and leukocytes and appears to play an important role in exocytosis through a currently unknown mechanism. Pleckstrin function is regulated by phosphorylation, which is thought to cause dissociation of pleckstrin dimers, thereby facilitating phosphoinositide interactions and membrane localization. Evidence also exists suggesting that phosphorylation causes a subtle conformational change in pleckstrin. Structural studies of pleckstrin have been initiated in order to characterize these structural changes and ultimately advance understanding of pleckstrin function. Here, the crystallization and preliminary X-ray diffraction analysis of a truncated version of pleckstrin consisting of the N-terminal PH domain, the protein kinase C phosphorylation sites and the DEP domain (NPHDEP) are reported. In addition, the oligomeric state and phospholipid-binding properties of NPHDEP were analyzed. This work demonstrates that NPHDEP behaves as a monomer in solution and suggests that all three pleckstrin domains contribute to the dimerization interface. Furthermore, based on the binding properties of NPHDEP, the C-terminal PH domain appears to increase the specificity of pleckstrin for phosphoinositides. This work represents a significant step towards determining the structure of pleckstrin.
pleckstrin; PH domain; DEP domain; protein kinase C
Crystals of a geranyl pyrophosphate methyltransferase in the biosynthetic pathway of the off-flavor terpenoid alcohol, 2-methylisoborneol were obtained in the absence and presence of cofactor, cofactor analog and substrate.
The biosynthetic pathway of the off-flavour terpenoid alcohol 2-methylisoborneol (2-MIB) requires geranyl pyrophosphate methyltransferase (GPPMT) to methylate GPP before the cyclization reaction. GPPMT is the first example of an S-adenosyl-l-methionine-dependent methyltransferase that acts on general intermediates such as geranyl pyrophosphate and farnesyl pyrophosphate in isoprenoid biosynthetic pathways. In this study, recombinant GPPMT was overproduced, purified and crystallized in the absence and presence of cofactor, cofactor analogue and substrate. Well diffracting crystals of apo GPPMT containing one molecule in the asymmetric unit were obtained and the structure of this form was solved by the molecular-replacement method. Two crystal forms of the tertiary complex with GPP and sinefungin were also obtained. Structure analysis of these crystals is currently under way in order to understand the enzyme reaction mechanism.
geranyl pyrophosphate methyltransferase; 2-methylisoborneol biosynthesis; Streptomyces lasaliensis
The effect of temperature during crystallization setup was analyzed and was found to influence precipitate formation and the crystal shape of the inactive E294A mutant of A. salmonicida ssp. achromogenes protease 1.
It is well known that protein crystallization is affected by several different parameters such as the composition of the reservoir solution, the protein concentration, the pH and the temperature. An effect of different temperatures during setup of crystallization experiments was observed for a metalloendopeptidase (AsaP1E294A). Spontaneous protein precipitation was reduced and the crystal shape could be improved by decreasing the temperature during crystallization setup.
crystal growth; temperature; precipitation; metalloendopeptidases; AsaP1