Triclosan (TCS) exposure impairs lipid metabolism in zebrafish embryos.

Triclosan (TCS) is an active antimicrobial ingredient used in many household products, such as skin creams and toothpaste. It is produced in high volumes, and humans are directly exposed to it and dispose it on a daily basis. TCS has been found to contaminate water worldwide. This study aimed to understand the potential developmental and metabolic abnormalities caused by TCS exposure by using zebrafish as the experimental model. Four developmental stages (70-85% epiboly, 10-12 somite, prim-5, and 5dpf) were selected to perform in situ hybridization staining to investigate the effects of TCS on dorsal ventral patterning, segmentation, brain development, and organ formation. Results showed, in terms of developmental toxicology, that neither phenotypic nor molecular changes were found after 5 days of 250µg/L TCS exposure. However, such dosage of TCS exposure resulted in lipid droplet accumulation in the yolk sac, which might due to the deregulated mRNA expression level of beta-oxidation transcripts. This study showed that 250µg/L TCS exposure does not affect normal embryogenesis or organogenesis; however, there are concerns regarding possible impairment of lipid metabolism.

Evaluation of nephrotoxic effects of aristolochic acid on zebrafish (Danio rerio) larvae.

To analyze the toxic effects of aristolochic acid (AA) on developed kidneys in zebrafish larvae, zebrafish at 3 days postfertilization were treated with various concentrations of AA for 24 h before the status of kidney injury was investigated from several points of view. It was found that 21% of the larvae treated with 10 µmoL/L AA exhibited evident periocular edema. When the concentrations of AA were increased to 20 and 40 µmoL/L, defect in the cardiovascular system characterized by slow heart beat and blood flow was seen coupled with periocular edema. Creatinine in the whole larval tissue determined by liquid chromatography-mass spectrometry/mass spectrometry exhibited dramatic increase in the treated groups in a dose-dependent manner within a certain range of doses. Several evident protein bands were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in supernatant of the treated larvae, indicating leakage of glomerular filtration barrier. Results of quantitative polymerase chain reaction show that the messenger RNA expression of nephrin in the 20 and 40 µmoL/L AA-treated groups decreased to 0.58 ± 0.062 and 0.37 ± 0.075-folds of the control, respectively. Kidney damage was further confirmed by the histological changes in paraffin sections of treated larvae, for example, cystic glomeruli and disorganized epithelia cells of pronephric tubules. Our results revealed that AA exerted toxic effects on developed kidney of zebrafish larvae in a dose-dependent manner and podocyte dysfunction may be involved in the kidney injury and proteinuria.

Crystallization and preliminary X-ray diffraction analysis of the 10 kDa C-terminal subdomain of 70 kDa heat-shock cognate protein.

The 70 kDa heat-shock cognate protein (Hsc70) is a cytosolic molecular chaperone. It is composed of a 44 kDa N-terminal nucleotide-binding domain, an 18 kDa peptide-binding subdomain and a 10 kDa C-terminal subdomain. Single crystals of recombinant 10 kDa subdomain of rat Hsc70 have been obtained using ammonium sulfate as a precipitant at room temperature. The crystals diffract beyond 3.5 A using a synchrotron-radiation source at a wavelength of 1.0 A. The crystals belong to the hexagonal space group P6(1)22 or P6(5)22, with unit-cell parameters a = b = 119.0, c = 166.4 A.

Boundary sequences of the NADPH oxidase p67(phox) C-terminal SH3 domain play on its specificity.

SH3 domains are found in many signal transduction proteins where they mediate protein-protein binding by recognizing specific peptides rich in proline. Based on the analysis of sequence alignment data, the NADPH oxidase p67(phox) C-terminal SH3 domain possesses a typical compact beta-barrel consisting of five beta-strands arranged in two antiparallel beta-sheets of three and two beta-strands. Multiple amino acid substitutions were made at beta e and its flanking residues to determine the role of the boundary sequences in binding activity and conformational specificity of the domain. Analysis of amino acid P55 indicated that all mutants were completely abolished in their binding activities. The substitution of F58 with Y58 showed no effect of the binding, whereas substitution with stop codon abolished activity. Furthermore, when amino acid V59 was substituted with stop codon, activity was also completely abolished. Substitution of E60 with stop codon showed no effect of binding. Moreover, our data show that V59 particularly could not be replaced by Leu. Taken together, these data suggest that V59 may not only contribute the exact boundary site but also play on the specificity for protein-protein interactions in phagocyte NADPH oxidase.

The effect of mutating arginine-469 on the substrate binding and refolding activities of 70-kDa heat shock cognate protein.

On the basis of the X-ray structure of DnaK, we obtained an energy-minimized model for the C-terminal domain of rat 70-kDa heat shock cognate protein (hsc70). The model suggests that Arg-469 may play an important role in maintaining the substrate-bound conformation of hsc70. To verify this hypothesis, we substituted cysteine for Arg-469 and generated the hsc70(R469C) mutant. Compared to the wild-type hsc70, the mutant was more accessible to cleavage by endopeptidase Lys-C, implying that the overall structure of hsc70(R469C) is relatively loose. Moreover, hsc70(R469C) did not form tightly associated complexes with S-carboxymethyl-alpha-lactalbumin, an unfolded protein. The amount of heptapeptide FYQLALT bound to hsc70(R469C) was also decreased as determined by gel filtration. Thus, the affinity of hsc70(R469C) for polypeptide substrates is reduced. In the presence of DnaJ, the capability of hsc70(R469C) to refold the denatured luciferase was decreased by 50%. Therefore, for hsc70, reduction in affinity for substrates may affect its DnaJ-dependent refolding activity.

Enhanced expression and stable transmission of transgenes flanked by inverted terminal repeats from adeno-associated virus in zebrafish.

Mosaic expression of transgenes in the F0 generation severely hinders the study of transient expression in transgenic fish. To avoid mosaicism, enhanced green fluorescent protein (EGFP) gene cassettes were constructed and introduced into one-celled zebrafish embryos. These EGFP gene cassettes were flanked by inverted terminal repeats (ITRs) from adeno-associated virus (AAV) and driven by zebrafish alpha-actin (palpha-actin-EGFP-ITR) or medaka beta-actin promoters (pbeta-actin-EGFP-ITR). EGFP was expressed specifically and uniformly in the skeletal muscle of 56% +/- 8% of the palpha-actin-EGFP-ITR-injected survivors and in the entire body of 1.3% +/- 0.8% of the pbeta-actin-EGFP-ITR-injected survivors. Uniform transient expression never occurred in zebrafish embryos injected with EGFP genes that were not flanked by AAV-ITRs. In the F0 generation, uniformly distributed EGFP could mimic the stable expression in transgenic lines early in development. We established five transgenic lines derived from palpha-actin-EGFP-ITR-injected embryos crossed with wild-type fish and 11 transgenic lines derived from pbeta-actin-EGFP-ITR-injected embryos crossed with wild-type fish. None of these transgenic lines failed to express the transgene, a result confirmed by polymerase chain reaction analysis. Stable mendelian transmission of the transgenes was achieved in both alpha-actin and beta-actin transgenic lines without changing the patterns of expression and integration. Progeny inheritance test and Southern blot analysis results strongly suggest that transgenes flanked by AAV-ITRs were integrated randomly into the genome at a single locus with a concatamerized multiplier. Thus, incorporating AAV-ITRs into transgenes results in uniform gene expression in the F0 generation and stable transmission of transgenes in zebrafish.

The crystal structure of a novel mammalian lectin, Ym1, suggests a saccharide binding site.

Ym1, a secretory protein synthesized by activated murine peritoneal macrophages, is a novel mammalian lectin with a binding specificity to GlcN. Lectins are responsible for carbohydrate recognition and for mediating cell-cell and cell-extracellular matrix interactions in microbes, plants, and animals. Glycosaminoglycan heparin/heparan sulfate binding ability was also detected in Ym1. We report here the three-dimensional structure of Ym1 at 2.5-A resolution by x-ray crystallography. The crystal structure of Ym1 consists of two globular domains, a beta/alpha triose-phosphate isomerase barrel domain and a small alpha + beta folding domain. A notable electron density of sugar is detected in the Ym1 crystal structure. The saccharide is located inside the triose-phosphate isomerase domain at the COOH terminal end of the beta-strands. Both hydrophilic and hydrophobic interactions are noted in the sugar-binding site in Ym1. Despite the fact that Ym1 is not a chitinase, structurally, Ym1 shares significant homology with chitinase A of Serratia marcescens. Ym1 and chitinase A have a similar carbohydrate binding cleft. This study provides new structure information, which will lead to better understanding of the biological significance of Ym1 and its putative gene members.

Tyr115, gln165 and trp209 contribute to the 1, 2-epoxy-3-(p-nitrophenoxy)propane-conjugating activity of glutathione S-transferase cGSTM1-1.

We investigated the epoxidase activity of a class mu glutathione S-transferase (cGSTM1-1), using 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) as substrate. Trp209 on the C-terminal tail, Arg107 on the alpha4 helix, Asp161 and Gln165 on the alpha6 helix of cGSTM1-1 were selected for mutagenesis and kinetic studies. A hydrophobic side-chain at residue 209 is needed for the epoxidase activity of cGSTM1-1. Replacing Trp209 with histidine, isoleucine or proline resulted in a fivefold to 28-fold decrease in the k(cat)(app) of the enzyme, while a modest 25 % decrease in the k(cat)(app) was observed for the W209F mutant. The rGSTM1-1 enzyme has serine at the correponding position. The k(cat)(app) of the S209W mutant is 2. 5-fold higher than that of the wild-type rGSTM1-1. A charged residue is needed at position 107 of cGSTM1-1. The K(m)(app)(GSH) of the R107L mutant is 38-fold lower than that of the wild-type enzyme. On the contrary, the R107E mutant has a K(m)(app)(GSH) and a k(cat)(app) that are 11-fold and 35 % lower than those of the wild-type cGSTM1-1. The substitutions of Gln165 with Glu or Leu have minimal effect on the affinity of the mutants towards GSH or EPNP. However, a discernible reduction in k(cat)(app) was observed. Asp161 is involved in maintaining the structural integrity of the enzyme. The K(m)(app)(GSH) of the D161L mutant is 616-fold higher than that of the wild-type enzyme. In the hydrogen/deuterium exchange experiments, this mutant has the highest level of deuteration among all the proteins tested. We also elucidated the structure of cGSTM1-1 co-crystallized with the glutathionyl-conjugated 1, 2-epoxy-3-(p-nitrophenoxy)propane (EPNP) at 2.8 A resolution. The product found in the active site was 1-hydroxy-2-(S-glutathionyl)-3-(p-nitrophenoxy)propane, instead of the conventional 2-hydroxy isomer. The EPNP moiety orients towards Arg107 and Gln165 in dimer AB, and protrudes into a hydrophobic region formed by the loop connecting beta1 and alpha1 and part of the C-terminal tail in dimer CD. The phenoxyl ring forms strong ring stacking with the Trp209 side-chain in dimer CD. We hypothesize that these two conformations represent the EPNP moiety close to the initial and final stages of the reaction mechanism, respectively.

The crystal structure of phosphoglucose isomerase/autocrine motility factor/neuroleukin complexed with its carbohydrate phosphate inhibitors suggests its substrate/receptor recognition.

Phosphoglucose isomerase catalyzes the reversible isomerization of glucose 6-phosphate to fructose 6-phosphate. In addition, phosphoglucose isomerase has been shown to have functions equivalent to neuroleukin, autocrine motility factor, and maturation factor. Here we present the crystal structures of phosphoglucose isomerase complexed with 5-phospho-D-arabinonate and N-bromoacetylethanolamine phosphate at 2.5- and 2.3-A resolution, respectively. The inhibitors bind to a region within the domains' interface and interact with a histidine residue (His(306)) from the other subunit. We also demonstrated that the inhibitors not only affect the enzymatic activity of phosphoglucose isomerase, but can also inhibit the autocrine motility factor-induced cell motility of CT-26 mouse colon tumor cells. These results indicate that the substrate and the receptor binding sites of phosphoglucose isomerase and autocrine motility factor are located within close proximity to each other. Based on these two complex structures, together with biological and biochemical results, we propose a possible isomerization mechanism for phosphoglucose isomerase.

Probing the location and function of the conserved histidine residue of phosphoglucose isomerase by using an active site directed inhibitor N-bromoacetylethanolamine phosphate.

Phosphoglucose isomerase (EC 5.3.1.9) catalyzes the interconversion of D-glucopyranose-6-phosphate and D-fructofuranose-6-phosphate by promoting an intrahydrogen transfer between C1 and C2. A conserved histidine exists throughout all phosphoglucose isomerases and was hypothesized to be the base catalyzing the isomerization reaction. In the present study, this conserved histidine, His311, of the enzyme from Bacillus stearothermophilus was subjected to mutational analysis, and the mutational effect on the inactivation kinetics by N-bromoacetylethanolamine phosphate was investigated. The substitution of His311 with alanine, asparagine, or glutamine resulted in the decrease of activity, in k(cat)/K(M), by a factor of 10(3), indicating the importance of this residue. N-bromoacetylethanolamine phosphate inactivated irreversibly the activity of wild-type phosphoglucose isomerase; however, His311 --> Ala became resistant to this inhibitor, indicating that His311 is located in the active site and is responsible for the inactivation of the enzyme by this active site-directed inhibitor. The pKa of His311 was estimated to be 6.31 according to the pH dependence of the inactivation. The proximity of this value with the pKa value of 6.35, determined from the pH dependence of k(cat)/K(M), supports a role of His311 as a general base in the catalysis.

Specific interaction of the 70-kDa heat shock cognate protein with the tetratricopeptide repeats.

Using a yeast two-hybrid system with the 70-kDa heat shock cognate protein (hsc70) or its C-terminal 30-kDa domain as baits, we isolated several proteins interacting with hsc70, including Hip/p48 and p60/Hop. Both are known to interact with hsc70. Except for Hip/p48, all of the proteins that we isolated interact with the 30-kDa domain. Moreover, the EEVD motif at the C terminus of the 30-kDa domain appears essential for this interaction. Sequence analysis of these hsc70-interacting proteins reveals that they all contain tetratricopeptide repeats. Using deletion mutants of these proteins, we demonstrated either by two-hybrid or in vitro binding assays that the tetratricopeptide repeat domains in these proteins are necessary and sufficient for mediating the interaction with hsc70.

The crystal structure of a multifunctional protein: phosphoglucose isomerase/autocrine motility factor/neuroleukin.

Phosphoglucose isomerase (PGI) plays a central role in both the glycolysis and the gluconeogenesis pathways. We present here the complete crystal structure of PGI from Bacillus stearothermophilus at 2.3-A resolution. We show that PGI has cell-motility-stimulating activity on mouse colon cancer cells similar to that of endogenous autocrine motility factor (AMF). PGI can also enhance neurite outgrowth on neuronal progenitor cells similar to that observed for neuroleukin. The results confirm that PGI is neuroleukin and AMF. PGI has an open twisted alpha/beta structural motif consisting of two globular domains and two protruding parts. Based on this substrate-free structure, together with the previously published biological, biochemical, and modeling results, we postulate a possible substrate-binding site that is located within the domains' interface for PGI and AMF. In addition, the structure provides evidence suggesting that the top part of the large domain together with one of the protruding loops might participate in inducing the neurotrophic activity.

The structure of glutamine-binding protein complexed with glutamine at 1.94 A resolution: comparisons with other amino acid binding proteins.

The crystal structure of the glutamine-binding protein (GlnBP) complexed with its ligand (Gln) was determined and refined to 1.94 A resolution. This ellipsoidal protein has two globular domains and is approximately 52 Ax40 Ax35 A in size. The glutamine ligand is located in the cleft between the two domains and stablized by hydrogen bondings and ionic interactions with Asp10, Gly68, Thr70, Ala67, Asp157, Arg75, Lys115, Gly119 and His156. The aliphatic portion of the glutamine ligand is sandwiched in a hydrophobic pocket formed between Phe13 and Phe50 and has 21 van der Waals contacts with GlnBP. Lys115 and His156, that are unique to GlnBP among amino acid binding proteins, apparently contribute to the ligand binding specificity of GlnBP. Asp10 is within 3 A of Lys115. These two residues are over 10 A apart in the ligand-free form of the GlnBP. In addition, GlnBP-Gln exhibits a large-scale movement of the two hinges connecting the two globular domains upon ligand binding. The most significant changes are 41.1 degrees in the phi angle of Gly89 and 34.3 degrees in the psi angle of Glu181 from the first and the second hinge of the protein, respectively. Besides the original six hydrogen bonds, three extra hydrogen bonds can be observed between the two hinge strands upon ligand binding. A hydrogen bond network connects the large domain to the second hinge and a second hydrogen bond network coalesces the small domain to the same strand, both via interaction with the glutamine ligand. Although the two strands of the hinge connecting the domains do not directly participate in the ligand binding, Gln183 and Tyr185 from the second hinge may be involved in the cascade of the conformational change that is induced by ligand binding.

The three-dimensional structure of an avian class-mu glutathione S-transferase, cGSTM1-1 at 1.94 A resolution.

Glutathione S-transferase cGSTM1-1, an avian class-mu enzyme with high sequence identity with rGSTM3-3, was expressed heterologously in Escherichia coli. The three-dimensional structure of this protein that co-crystallized with an inhibitor, S-hexylglutathione, was determined by the molecular replacement method and refined to 1.94 A resolution. The three-dimensional structure and the folding topology of the dimeric cGSTM1-1 closely resembles those of other class-mu GSTs. The bound inhibitor, S-hexylglutathione, orients in disparate directions in the two subunits. The combined space occupied by the hexyl moiety of the inhibitors overlaps with that reported for rGSTM1-1 co-crystallized with (9 S,10 S)-9-(S-glutathionyl)-10-hydroxy-9,10-dihydrophenanthrene. Conformational differences at a flexible loop (residue 35 to 40) were also observed between the crystal structures of cGSTM1-1 and rGSTM1-1.cGSTM1-1 has the highest epoxidase activity among all the class-mu enzymes reported. Tyr115, has been identified as a residue that participates in the epoxidase activity of class-mu glutathione S-transferase and is conserved in cGSTM1-1. The epoxidase and trans-4-phenyl-3-buten-2-one conjugating activity of cGSTM1-1 are decreased drastically but not abolished by replacing Tyr115 with phenylalanine. The specificity constant of the cGSTM1-1(Y115F) mutant, with 1-chloro-2,4-dinitrobenzene as substrate, is 15-fold higher than that of the wild-type enzyme.

The first structure of an aldehyde dehydrogenase reveals novel interactions between NAD and the Rossmann fold.

The first structure of an aldehyde dehydrogenase (ALDH) is described at 2.6 A resolution. Each subunit of the dimeric enzyme contains an NAD-binding domain, a catalytic domain and a bridging domain. At the interface of these domains is a 15 A long funnel-shaped passage with a 6 x 12 A opening leading to a putative catalytic pocket. A new mode of NAD binding, which differs substantially from the classic beta-alpha-beta binding mode associated with the 'Rossmann fold', is observed which we term the beta-alpha,beta mode. Sequence comparisons of the class 3 ALDH with other ALDHs indicate a similar polypeptide fold, novel NAD-binding mode and catalytic site for this family. A mechanism for enzymatic specificity and activity is postulated.

Crystal structure of cardiotoxin V from Taiwan cobra venom: pH-dependent conformational change and a novel membrane-binding motif identified in the three-finger loops of P-type cardiotoxin.

The crystal structure of cardiotoxin V from Taiwan cobra venom (CTX A5) has been solved at pH 8.5 and refined to an R-factor of 20.7% for 7013 reflections [>2sigma(F)] between 8- and 2.19-A resolution. The refined model shows that CTX A5 exists as a dimer. The assembly consists of 974 non-hydrogen atoms from 124 residues and 73 water molecules. The global monomeric structure is similar to that determined by NMR at pH 3.7, characterized by a core formed by two beta-sheets connected with three-finger loops. However, local conformational differences are detected in two functionally important regions, loops I and II. A disparity between the NMR and X-ray structure of CTX A5 is detected near the tip of loop I and can be attributed to the difference in the protonation state of His4 at different pH, resulting in a reorientation of the His4 imidazole ring. A concerted motion of amino acid side chains located near His4 is detected and possibly contributes to the pH-dependent binding ability of CTX A5 to phospholipid model membranes. The second difference, detected at the tip of loop II, is due to the hydrophobic contact between CTX dimers in the crystal packing and the interaction of water molecules with amino acid residues in the loop II region of the CTX containing Pro31 (P-type CTX). This interaction forces loop II into a more rigid omega shape bridging the main chain at positions 27 and 34, contradictory to the flexible, tapering shape detected by NMR. Thus, a novel continuous hydrophobic column capable of binding to and possibly penetrating the membrane lipid bilayer is formed by the tips of the three-finger loops. In this respect, the X-ray crystal structure of CTX A5 may represent the CTX structure in the membrane-binding mode.

Expression, purification, and crystallization of two isozymes of 6-phosphoglucose isomerase of Bacillus stearothermophilus.

Two isozymes of 6-phosphoglucose isomerase (phosphoglucose isomerase A & phosphoglucose isomerase B), isolated from Bacillus stearothermophilus, have been overexpressed in Escherichia coli strain DF2145 and purified to homogeneity. Crystals of both isozymes have been obtained by the vapor diffusion method. The crystals of phosphoglucose isomerase A have unit cell dimensions a = b = 132.0 A, c = 183.6 A, and diffract to about 2.8 A resolution. An analysis of the reflection data indicates that the crystal system is hexagonal, space group P6122 or P6522. The crystals of phosphoglucose isomerase B complexed with 6-phosphogluconate belong to the orthorhombic space group I222 (or I212121), with cell dimensions a = 75.1 A, b = 95.7 A, c = 171.5 A, and diffract to a resolution of 2.3 A. These crystals promise to yield more detail for the substrate recognition and higher resolution structures of 6-phosphoglucose isomerase.

A novel neurotoxin, cobrotoxin b, from Naja naja atra (Taiwan cobra) venom: purification, characterization, and gene organization.

A novel neurotoxin, cobrotoxin b, was isolated from Naja naja atra (Taiwan cobra) venom by successive chromatographies on gel filtration and SP-Sephadex C-25 columns. The yield of this novel toxin was 5% of that of cobrotoxin from the same venom. Its neurotoxicity determined as the inhibition of acetylcholine-induced muscle contractions was approximately 50% of that of cobrotoxin. Cobrotoxin b consists of 61 amino acid residues including 8 cysteine residues. Moreover, there are 12 amino acid substitutions between cobrotoxin b and cobrotoxin. The genomic DNA, with a size of 2,386bp, encoding the precursor of cobrotoxin b was isolated from the liver of N. naja atra. The gene consists of three exons separated by two introns. This exon/intron structure is essentially the same as that reported for the cobrotoxin gene. Moreover, the nucleotide sequences of the two neurotoxin genes exhibit 92% identity. These results highly suggest that the cobrotoxin b and cobrotoxin genes are derived from a common ancestor. Comparative analyses of cobrotoxin b and cobrotoxin precursors showed that the protein-coding regions of the exons are more diverse than introns, except for in the signal peptide domain. This indicates that the protein-coding regions may have arised via accelerated evolution. BLAST searches for sequence similarity in the GeneBank databases showed that intron 1 of the cobrotoxin b and cobrotoxin genes encodes a small nucleolar RNA (snoRNA). However, the snoRNA gene is absent from the gene encoding the Laticauda semifasciata erabutoxin c precursor (L. semifasciata and N. naja atra are sea and land snakes, respectively). Since previous studies suggested the potential mobility of snoRNA genes during evolution, we propose that intron insertions or deletions of snoRNA genes occurred with the evolutionary divergence between the sea snake and land snake neurotoxins.

Neuronal type information encoded in the basic-helix-loop-helix domain of proneural genes.

The proneural genes encode basic-helix-loop-helix (bHLH) proteins and promote the formation of distinct types of sensory organs. In Drosophila, two sets of proneural genes, atonal (ato) and members of the achaete-scute complex (ASC), are required for the formation of chordotonal (ch) organs and external sensory (es) organs, respectively. We assayed the production of sensory organs in transgenic flies expressing chimeric genes of ato and scute (sc), a member of ASC, and found that the information that specifies ch organs resides in the bHLH domain of ato; chimeras containing the b domain of ato and the HLH domain of sc also induced ch organ formation, but to a lesser extent than those containing the bHLH domain of ato. The b domains of ato and sc differ in seven residues. Mutations of these seven residues in the b domain of ato suggest that most or perhaps all of these residues are required for induction of ch organs. None of these seven residues is predicted to contact DNA directly by computer simulation using the structure of the myogenic factor MyoD as a model, implying that interaction of ato with other cofactors is likely to be involved in neuronal type specification.

The crystal structure of glutamine-binding protein from Escherichia coli.

The crystal structure of the glutamine-binding protein (GlnBP) from Escherichia coli in a ligand-free "open" conformational state has been determined by isomorphous replacement methods and refined to an R-value of 21.4% at 2.3 A resolution. There are two molecules in the asymmetric unit, related by pseudo 4-fold screw symmetry. The refined model consists of 3587 non-hydrogen atoms from 440 residues (two monomers), and 159 water molecules. The structure has root-mean-square deviations of 0.013 A from "ideal" bond lengths and 1.5 degrees from "ideal" bond angles. The GlnBP molecule has overall dimensions of approximately 60 A x 40 A x 35 A and is made up of two domains (termed large and small), which exhibit a similar supersecondary structure, linked by two antiparallel beta-strands. The small domain contains three alpha-helices and four parallel and one antiparallel beta-strands. The large domain is similar to the small domain but contains two additional alpha-helices and three more short antiparallel beta-strands. A comparison of the secondary structural motifs of GlnBP with those of other periplasmic binding proteins is discussed. A model of the "closed form" GlnBP-Gln complex has been proposed based on the crystal structures of the histidine-binding protein-His complex and "open form" GlnBP. This model has been successfully used as a search model in the crystal structure determination of the "closed form" GlnBP-Gln complex by molecular replacement methods. The model agrees remarkably well with the crystal structure of the Gln-GlnBP complex with root-mean-square deviation of 1.29 A. Our study shows that, at least in our case, it is possible to predict one conformational state of a periplasmic binding protein from another conformational state of the protein. The glutamine-binding pockets of the model and the crystal structure are compared and the modeling technique is described.