Crystal structure of ATP-binding subunit of an ABC transporter from Geobacillus kaustophilus.

The ATP binding cassette (ABC) transporters, represent one of the largest superfamilies of primary transporters, which are very essential for various biological functions. The crystal structure of ATP-binding subunit of an ABC transporter from Geobacillus kaustophilus has been determined at 1.77 Å resolution. The crystal structure revealed that the protomer has two thick arms, (arm I and II), which resemble 'L' shape. The ATP-binding pocket is located close to the end of arm I. ATP molecule is docked into the active site of the protein. The dimeric crystal structure of ATP-binding subunit of ABC transporter from G. kaustophilus has been compared with the previously reported crystal structure of ATP-binding subunit of ABC transporter from Salmonella typhimurium.

Crystal structure of product-bound complex of UDP-N-acetyl-d-mannosamine dehydrogenase from Pyrococcus horikoshii OT3.

UDP-N-acetyl-d-mannosamine dehydrogenase (UDP-d-ManNAcDH) belongs to UDP-glucose/GDP-mannose dehydrogenase family and catalyzes Uridine-diphospho-N-acetyl-d-mannosamine (UDP-d-ManNAc) to Uridine-diphospho-N-acetyl-d-mannosaminuronic acid (UDP-d-ManNAcA) through twofold oxidation of NAD(+). In order to reveal the structural features of the Pyrococcus horikoshii UDP-d-ManNAcADH, we have determined the crystal structure of the product-bound enzyme by X-ray diffraction to resolution of 1.55Å. The protomer folds into three distinct domains; nucleotide binding domain (NBD), substrate binding domain (SBD) and oligomerization domain (OD, involved in the dimerization). The clear electron density of the UDP-d-ManNAcA is observed and the residues binding are identified for the first time. Crystal structures reveal a tight dimeric polymer chains with product-bound in all the structures. The catalytic residues Cys258 and Lys204 are conserved. The Cys258 acts as catalytic nucleophile and Lys204 as acid/base catalyst. The product is directly interacts with residues Arg211, Thr249, Arg244, Gly255, Arg289, Lys319 and Arg398. In addition, the structural parameters responsible for thermostability and oligomerization of the three dimensional structure are analyzed.

A planning target volume margin formula for hypofractionated intracranial stereotactic radiotherapy under cone beam CT image guidance with a six-degrees-of-freedom robotic couch and a mouthpiece-assisted mask system: a preliminary study.

OBJECTIVE: A planning target volume (PTV) margin formula for hypofractionated intracranial stereotactic radiotherapy (SRT) has been proposed under cone beam CT (CBCT) image guidance with a six-degrees-of-freedom (6-DOF) robotic couch. METHODS: CBCT-based registration using a 6-DOF couch reportedly led to negligibly small systematic positioning errors, suggesting that each in-treatment positioning error during the treatment courses for the patients employing this combination was predominantly caused by a random gaussian process. Under this assumption, an anisotropic PTV margin for each axis was formulated based on a gaussian distribution model. 19 patients with intracranial lesions who underwent additional post-treatment CBCT were consecutively selected, to whom stereotactic hypofractionated radiotherapy was delivered by a linear accelerator equipped with a CBCT imager, a 6-DOF couch and a mouthpiece-assisted mask system. Time-averaged patient-positioning errors during treatment were estimated by comparing the post-treatment CBCT with the reference planning CT images. RESULTS: It was suggested that each histogram of the in-treatment positioning error in each axis would approach each single gaussian distribution with a mean of zero. The calculated PTV margins in the x, y and z directions were 0.97, 1.30 and 0.88 mm, respectively. CONCLUSION: The empirical isotropic PTV margin of 2 mm used in our facility for intracranial SRT was consistent with the margin calculated by the proposed gaussian model. ADVANCES IN KNOWLEDGE: We have proposed a PTV margin formula for hypofractionated intracranial SRT under CBCT image guidance with a 6-DOF robotic couch.

Crystal structure of the Holliday junction migration motor protein RuvB from Thermus thermophilus HB8.

We report here the crystal structure of the RuvB motor protein from Thermus thermophilus HB8, which drives branch migration of the Holliday junction during homologous recombination. RuvB has a crescent-like architecture consisting of three consecutive domains, the first two of which are involved in ATP binding and hydrolysis. DNA is likely to interact with a large basic cleft, which encompasses the ATP-binding pocket and domain boundaries, whereas the junction-recognition protein RuvA may bind a flexible beta-hairpin protruding from the N-terminal domain. The structures of two subunits, related by a noncrystallographic pseudo-2-fold axis, imply that conformational changes of motor protein coupled with ATP hydrolysis may reflect motility essential for its translocation around double-stranded DNA.

Structural basis of glutamate recognition by a dimeric metabotropic glutamate receptor.

The metabotropic glutamate receptors (mGluRs) are key receptors in the modulation of excitatory synaptic transmission in the central nervous system. Here we have determined three different crystal structures of the extracellular ligand-binding region of mGluR1--in a complex with glutamate and in two unliganded forms. They all showed disulphide-linked homodimers, whose 'active' and 'resting' conformations are modulated through the dimeric interface by a packed alpha-helical structure. The bi-lobed protomer architectures flexibly change their domain arrangements to form an 'open' or 'closed' conformation. The structures imply that glutamate binding stabilizes both the 'active' dimer and the 'closed' protomer in dynamic equilibrium. Movements of the four domains in the dimer are likely to affect the separation of the transmembrane and intracellular regions, and thereby activate the receptor. This scheme in the initial receptor activation could be applied generally to G-protein-coupled neurotransmitter receptors that possess extracellular ligand-binding sites.

Purification, crystallization and preliminary X-ray analysis of a complex between granulocyte colony-stimulating factor and its soluble receptor.

Crystals of the complex between granulocyte colony-stimulating factor and its soluble receptor were obtained by a vapour-diffusion method using ammonium sulfate as a precipitant. Addition of 1, 4-dioxane was critical in order to grow the crystals to sufficient sizes. Cryoprotection was essential in order to collect diffraction data at atomic resolution. Two kinds of crystal forms were obtained depending on the cryoprotectants. In a cryosolvent with the same salt concentration as in the crystallization conditions, the crystal belonged to the space group I4(1)22. At higher salt concentrations, the crystal was converted to a different space group P4(1)2(1)2 (P4(3)2(1)2) with the same unit-cell parameters.

An electrospray-ionization mass spectrometry analysis of the pH-dependent dissociation and denaturation processes of a heterodimeric protein.

Electrospray ionization mass spectrometry (ESI-MS) was applied to the analysis of the dissociation and denaturation processes of a heterodimeric yeast killer toxin SMKT. The two distinct subunits of SMKT noncovalently associate under acidic conditions, but become dissociated and denatured under neutral and basic conditions. In order to understand the unique pH-dependent denaturation mechanism of this protein, a pH titration was performed by utilizing ESI-MS. The molecular ions of the heterodimer which possesses the highly ordered structure, were mainly observed below pH 4.6. However, the two subunits immediately dissociated at this pH. The spectra measured with various settings of the mass spectrometer indirectly demonstrated that the pH-dependent dissociation occurs in the liquid phase. The current result as well as the three-dimensional structure of SMKT suggest that the deprotonation of a specific carboxyl group triggers a cooperative dissociation process of this protein. In conclusion, the pH titration of a protein by ESI-MS is particularly effective, when the unfolding process or the biological function of the protein is related to the interaction with other molecules.

Atomic structure of the GCSF-receptor complex showing a new cytokine-receptor recognition scheme.

Granulocyte colony-stimulating factor (GCSF) is the principal growth factor regulating the maturation, proliferation and differentiation of the precursor cells of neutrophilic granulocytes and is used to treat neutropenia. GCSF is a member of the long-chain subtype of the class 1 cytokine superfamily, which includes growth hormone, erythropoietin, interleukin 6 and oncostatin M. Here we have determined the crystal structure of GCSF complexed to the BN-BC domains, the principal ligand-binding region of the GCSF receptor (GCSFR). The two receptor domains form a complex in a 2:2 ratio with the ligand, with a non-crystallographic pseudo-twofold axis through primarily the interdomain region and secondarily the BC domain. This structural view of a gp130-type receptor-ligand complex presents a new molecular basis for cytokine-receptor recognition.

Crystallographic and functional studies of very short patch repair endonuclease.

Vsr endonuclease plays a crucial role in the repair of TG mismatched base pairs, which are generated by the spontaneous degradation of methylated cytidines; Vsr recognizes the mismatched base pair and cleaves the phosphate backbone 5' to the thymidine. We have determined the crystal structure of a truncated form of this endonuclease at 1.8 A resolution. The protein contains one structural zinc-binding module. Unexpectedly, its overall topology resembles members of the type II restriction endonuclease family. Subsequent mutational and biochemical analyses showed that certain elements in the catalytic site are also conserved. However, the identification of a critical histidine and evidence of an active site metal-binding coordination that is novel to endonucleases indicate a distinct catalytic mechanism.

Crystal structure of rat Bcl-xL. Implications for the function of the Bcl-2 protein family.

Bcl-xL is a member of the Bcl-2 protein family, which regulates apoptosis. Preparation of recombinant rat Bcl-xL yielded two forms, one deamidated at -Asn-Gly- sequences to produce isoaspartates and the other not deamidated. The crystal structures of the two forms show that they both adopt an essentially identical backbone structure which resembles the fold of human Bcl-xL: three layers of two alpha-helices each, capped at one end by two short helices. Both forms have a long disordered region, which contains the potential deamidation sites. The molecular structure exhibits a low level of interhelical interactions, the presence of three cavities, and a notable hydrophobic cleft surrounded by walls rich in basic residues. These unique structural features may be favorable for its accommodation into membranes or for possible rearrangement to modulate homo-/heterodimerization. Homology modeling of Bcl-2 and Bax, based on the Bcl-xL structure, suggests that Bax has the strongest potential for membrane insertion. Furthermore, we found a possible interface for interaction with non-Bcl-2 family member proteins, such as CED-4 homologues.

Circular dichroism analysis of the interaction between the alpha and beta subunits in a killer toxin produced by a halotolerant yeast, Pichia farinosa.

SMK toxin is a killer toxin produced by a halotolerant yeast, Pichia farinosa. It is a heterodimer consisting of alpha (63 aa) and beta (77 aa) subunits, between which no disulfide bond exists. The two subunits interact tightly with each other below pH 5. However, the subunits dissociate under neutral conditions, resulting in the aggregation of the alpha subunit and the concomitant loss of killer activity. CD spectral measurements showed that the secondary structure of the SMK toxin changes drastically in the pH range 5.1-5.5 and that after the dissociation of the subunits, the soluble beta subunit alone cannot take any secondary structure. It was also shown that the concentration of NaCl does not affect the secondary structure of the SMK toxin.

The novel acidophilic structure of the killer toxin from halotolerant yeast demonstrates remarkable folding similarity with a fungal killer toxin.

BACKGROUND: Several strains of yeasts and fungi produce proteinous substances, termed killer toxins, which kill sensitive strains. The SMK toxin, secreted by the halotolerant yeast Pichia farinosa KK1 strain, uniquely exhibits its maximum killer activity under conditions of acidic pH and high salt concentration. The toxin is composed of two distinct subunits, alpha and beta, which tightly interact with each other under acidic conditions. However, they are easily dissociated under neutral conditions and lose the killer activity. The three-dimensional structure of the SMK toxin will provide a better understanding of the mechanism of toxicity of this protein and the cause of its unique pH-dependent stability. RESULTS: Two crystal structures of the SMK toxin have been determined at 1.8 A resolution in different ionic strength conditions. The two subunits, alpha and beta, are jointly folded into an ellipsoidal, single domain structure belonging to the alpha/beta-sandwich family. The folding topology of the SMK toxin is essentially the same as that of the fungal killer toxin, KP4. This shared topology contains two left-handed split betaalphabeta motifs, which are rare in the other proteins. Many acidic residues are clustered at the bottom of the SMK toxin molecule. Some of the carboxyl sidechains interact with each other through hydrogen bonds. The ionic strength difference induces no evident structural change of the SMK toxin except that, in the high ionic strength crystal, a number of sulfate ions are electrostatically bound near the basic residues which are also locally distributed at the bottom of the toxin molecule. CONCLUSIONS: The two killer toxins, SMK and KP4, share a unique folding topology which contains a rare structural motif. This observation may suggest that these toxins are evolutionally and/or functionally related. The pH-dependent stability of the SMK toxin is a result of the intensive interactions between the carboxyl groups. This finding is important for protein engineering, for instance, towards stabilization of the toxin molecule in a broader pH range. The present crystallographic study revealed that the structure of the SMK toxin itself is hardly affected by the ionic strength, implying that a high salt concentration affects the sensitivity of the cell against the toxin.

Crystallization and preliminary X-ray diffraction studies of a novel killer toxin from a halotolerant yeast Pichia farinosa.

A killer toxin from a halotolerant yeast, Pichia farinosa strain KK1, was crystallized at high- and low-salt concentrations. Crystals from the high-salt solution belonged to the tetragonal space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell dimensions of a = b = 81.10, c = 118.46 A. The low-salt solution provided crystals that belonged to the same space group, with nearly same cell dimensions. Preliminary diffraction studies showed that the intensity distributions are significantly different between the two crystals. Both types of crystals contained either two or three molecules per asymmetric unit. They diffracted X-rays beyond 2.0 A resolution and were stable to X-ray irradiation.

Pentacoordination of the heme iron of Arthromyces ramosus peroxidase shown by a 1.8 A resolution crystallographic study at pH 4.5.

In the presence of ammonium sulfate the absorption spectra of a peroxidase from the fungus Arthromyces ramosus (ARP) showed that the low-spin component increased as the pH increased from 6.0 to 9.0, whereas in its absence ARP remained in the high-spin state in the pH range investigated. The crystal structure of ARP at pH 4.5 in the presence of ammonium sulfate at 1.8 A resolution showed that the electron density at the 6th position of the heme iron seen at pH 7.5 had disappeared and that the iron atom deviated markedly from the heme plane. These observations strongly suggest that under physiological conditions the heme of ARP is in the pentacoordinated high-spin state and that at a high pH the heme iron is able to bind ammonia, forming the low-spin state. The location of the water molecule at the distal side of the heme in peroxidases is also discussed.

Crystal structures of cyanide- and triiodide-bound forms of Arthromyces ramosus peroxidase at different pH values. Perturbations of active site residues and their implication in enzyme catalysis.

The structures of the cyanide and triiodide complexes of Arthromyces ramosus peroxidase (ARP) at different pH values were investigated by x-ray crystallography in order to examine the behavior of the invariant residues of arginine (Arg-52) and distal histidine (His-56) during the enzyme reaction as well as to provide the structural basis of the active site of peroxidase. The models of the cyanide complexes at pH 7.5, 5.0, and 4.0, respectively, were refined to the R-factors of 17.8, 17.8, and 18.5% using 7.0-1.6-A resolution data, and those of the triiodide complexes at pH 6.5 and 5.0 refined to 16.9 and 16.8% using 7.0-1.9-A resolution data. The structures of the cyanide complexes at pH 7.5, 5.0, and 4.0 are identical within experimental error. Cyanide ion bound to the heme in the bent conformation rather than in the tilt conformation. Upon cyanide ion binding, the N epsilon atom of His-56 moved toward the ion by rotation of the imidazole ring around the C beta-C gamma bond, but there was little conformational change in the remaining residues. The distance between the N epsilon atom of His-56 and the nitrogen atom of the cyanide suggests the presence of a hydrogen bond between them in the pH range investigated. In the triiodide complexes, one of the two triiodides bound to ARP was located at the distal side of the heme. When triiodide bound to ARP, unlike the rearrangement of the distal arginine of cytochrome c peroxidase that occurs on formation of the fluoride complex or compound I, the side chain of Arg-52 moved little. The conformation of the side chain of His-56, however, changed markedly. Conformational flexibility of His-56 appears to be a requisite for proton translocation from one oxygen atom to the other of HOO- by acid-base catalysis to produce compound I. The iron atom in each cyanide complex (low-spin ferric) is located in the heme plane, whereas in each triiodide complex (high-spin ferric) the iron atom is displaced from the plane about 0.2 A toward the proximal side.

Cloning, sequencing, and heterologous expression of a gene coding for Arthromyces ramosus peroxidase.

To understand the relationship between the structure and functions of the peroxidase of Arthromyces ramosus, a novel taxon of hyphomycete, and the evolutionary relationship of the A.ramosus peroxidase (ARP) with the other peroxidases, we isolated complementary and genomic DNA clones encoding ARP and characterized them. The sequence analyses of the ARP and cDNA coding for ARP showed that a mature ARP consists of 344 amino acids with a N-terminal pyroglutamic acid preceded by a signal peptide of 20 amino acid residues. The amino acid sequence of ARP was 99% identical to that of the peroxidase of Coprinus cinereus, a basidiomycete, and also had very high similarities (41-43% identity) to those of basidiomycetous lignin peroxidases, although we could find no lignin peroxidase activities for ARP when assayed with lignin model compounds. We could identified His184 and His56 as proximal and distal ligands to heme, respectively, and Arg52 as an essential Arg. Comparison of the sequences of complementary and genomic DNAs found that protein-encoding DNA is interrupted by 14 intervening sequences. The ARP cDNA was expressed in the yeast Saccharomyces cerevisiae under the promoter of the glyceraldehyde 3-phosphate dehydrogenase gene, yielding 0.02 units/ml of a secreted active peroxidase.

Acute pneumonitis presumed to be silicone embolism.

A 39-year-old housewife who underwent intramammary injections of a proprietary silicone fluid mixture showed clinical and novel transbronchial lung biopsy (TBLB) findings. She presented with complaints of progressive dyspnea, dry cough, and pleuritic chest pain 2 days after the last silicone injections. The chest X-ray and CT scan showed diffuse interstitial infiltrates. TBLB demonstrated translucent, presumably silicone globules embolized within the pulmonary capillaries. The documentation of intramammary injections, the clinical and radiographic features of acute pneumonitis, and the histopathologic evidence by TBLB, may support the causal relationship between illicit injections and the silicone embolism. We discuss the pathogenesis and urge that this potentially toxic source of pulmonary embolism be removed.

Crystal structure of the fungal peroxidase from Arthromyces ramosus at 1.9 A resolution. Structural comparisons with the lignin and cytochrome c peroxidases.

The crystal structure of the peroxidase (donor: H2O2 oxidoreductase, EC 1.11.1.7) from the hyphomycete Arthromyces ramosus (ARP) has been determined by the multiple isomorphous replacement method and refined by the simulated annealing method to a crystallographic R-factor of 17.4% for the 19,191 reflections with F > 2 sigma F between 7.0 and 1.9 A resolution. The model includes residues 9 to 344, the heme group, two N-acetylglucosamine residues, two calcium ions and 246 water molecules. The root-mean-square deviation of bond lengths from the ideal values is 0.02 A. The mean coordinate error is estimated as 0.2 A. The electron density of the glycine-rich region of the amino-terminal eight residues was invisible. ARP has ten major and two short alpha-helices and a few short beta-strands. The overall tertiary structure of ARP is similar to that of yeast cytochrome c peroxidase (CCP) and is particularly similar to that of the lignin peroxidase (LiP) from Phanerochaete chrysosporium. Relative to CCP, ARP and LiP each have an extension of approximately 40 residues at the carboxy terminus. All eight cysteine residues in ARP form disulfide bonds (C12:C24, C23:C293, C43:C129 and C257:C322). Two calcium sites are inaccessible to solvent. The four disulfide bonds and two calcium sites, which are lacking in CCP, are conserved in ARP and LiP. The bond from Asn304C to Ala305N in ARP is the site sensitive to proteases. An Asx turn present in the Asn303 to Ala305 segment appears to orient the side-chain of Asn304 to outward from the molecule, rendering it easily trappable by pockets of proteases. The proximal heme ligand is His184 in helix F (distance of N epsilon 2 ... Fe, 2.10 A), and one of several water molecules in the distal pocket of the heme bridges the iron atom and the N epsilon 2 of His56. The orientation of the imidazole ring of the distal histidine residue relative to the heme group in ARP differs significantly from that in LiP. The access channel to the distal side of the heme of ARP is markedly wider along the heme plane than that of LiP. Many of the amino acid residues that comprise the entrance of this channel differ for ARP and LiP. This may account for the differences in substrate specificity.