Structure and dynamics of the anticodon arm binding domain of Bacillus stearothermophilus Tyrosyl-tRNA synthetase

The structure of a recombinant protein, TyrRS(delta4), corresponding to the anticodon arm binding domain of Bacillus stearothermophilus tyrosyl-tRNA synthetase, has been solved, and its dynamics have been studied by nuclear magnetic resonance (NMR). It is the first structure described for such a domain of a tyrosyl-tRNA synthetase. It consists of a five-stranded beta sheet, packed against two alpha helices on one side and one alpha helix on the other side. A large part of the domain is structurally similar to other functionally unrelated RNA binding proteins. The basic residues known to be essential for tRNA binding and charging are exposed to the solvent on the same face of the molecule. The structure of TyrRS(delta4), together with previous mutagenesis data, allows one to delineate the region of interaction with tRNATyr.     

Associative properties of the Escherichiacoli galactose binding protein and maltose binding protein

The periplasmic galactose binding protein and maltose binding protein of $\text{Escherichia}\text{coli}$ are recovered mostly in dimeric form when purified, from osmotically-shocked bacteria, in the presence of protease inhibitors and 2-mercaptoethanol without dialysis and concentration of the shock fluid. The specific ligands, galactose (but not glucose) for galactose binding protein, and maltose for maltose binding protein, provoque the monomerisation of the dimeric native forms. These results are discussed in relation to the function of both binding proteins in transport and chemotaxis.     

Three-dimensional solution structure of Acanthamoeba profilin-I

We have determined a medium resolution three-dimensional solution structure of Acanthamoeba profilin-I by multidimensional nuclear magnetic resonance spectroscopy. This 13-kD actin binding protein consists of a five stranded antiparallel beta sheet flanked by NH2- and COOH-terminal helices on one face and by a third helix and a two stranded beta sheet on the other face. Data from actin-profilin cross- linking experiments and the localization of conserved residues between profilins in different phyla indicate that actin binding occurs on the molecular face occupied by the terminal helices. The other face of the molecule contains the residues that differ between Acanthamoeba profilins-I and II and may be important in determining the difference in polyphosphoinositide binding between these isoforms. This suggests that lipids and actin bind to different faces of the molecule.     

Structure of the nuclear factor ALY: insights into post-transcriptional regulatory and mRNA nuclear export processes

ALY is a ubiquitously expressed nuclear protein which interacts with proteins such as TAP that are involved in export of mRNA from the nucleus to the cytoplasm, as well as with proteins that bind the T cell receptor alpha gene enhancer. ALY has also been shown to bind mRNA and to co-localize in the nucleus with components of a multiprotein postsplicing complex that is deposited 20-24 nucleotides upstream of exon-exon junctions. ALY has a conserved RNA binding domain (RBD) flanked by Gly-Arg rich N-terminal and C-terminal sequences. We determined the solution structure of the RBD homology region in ALY by nuclear magnetic resonance methods. The RBD motif in ALY has a characteristic beta(1)alpha(1)beta(2)-beta(3)alpha(2)beta(4) fold, consisting of a beta sheet composed of four antiparallel beta strands and two alpha helices that pack on one face of the beta sheet. As in other RBD structures, the beta sheet has an exposed face with hydrophobic and charged residues that could modulate interactions with other molecules. The loop that connects beta strands 2 and 3 is in intermediate motion in the NMR time scale, which is also characteristic of other RBDs. This loop presents side chains close to the exposed surface of the beta sheet and is a primary candidate site for intermolecular interactions. The structure of the conserved RBD of ALY provides insight into the nature of interactions involving this multifunctional protein.     

Crystallographic evidence of a large ligand-induced hinge-twist motion between the two domains of the maltodextrin binding protein involved in active transport and chemotaxis.

The periplasmic maltodextrin binding protein of Escherichia coli serves as an initial receptor for the active transport of and chemotaxis toward maltooligosaccharides. The three-dimensional structure of the binding protein complexed with maltose has been previously reported [Spurlino, J. C., Lu, G.-Y., & Quiocho, F. A. (1991) J. Biol. Chem. 266, 5202-5219]. Here we report the structure of the unliganded form of the binding protein refined to 1.8-A resolution. This structure, combined with that for the liganded form, provides the first crystallographic evidence that a major ligand-induced conformational change occurs in a periplasmic binding protein. The unliganded structure shows a rigid-body "hinge-bending" between the two globular domains by approximately 35 degrees, relative to the maltose-bound structure, opening the sugar binding site groove located between the two domains. In addition, there is an 8 degrees twist of one domain relative to the other domain. The conformational changes observed between this structure and the maltose-bound structure are consistent with current models of maltose/maltodextrin transport and maltose chemotaxis and solidify a mechanism for receptor differentiation between the ligand-free and ligand-bound forms in signal transduction.     

Structure determination of histidine decarboxylase from Lactobacillus 30a at 3.0 A resolution

The crystal structure of histidine decarboxylase from Lactobacillus 30a has been determined by X-ray diffraction methods to a resolution of 3.0 A. This protein is a pyruvoyl-dependent enzyme that is formed by an unusual self-activation process. The structure was determined from an electron density map calculated using multiple isomorphous replacement phases from two heavy-atom derivatives and included contributions from anomalous scattering measurements. The final mean figure of merit was 0.79, based on 28,805 independent reflections. The molecule has an (alpha beta)6 subunit composition and crystallizes in the space group 14122 with a = b = 221.7 A and c = 107.1 A. There is one (alpha beta)3 half molecule per asymmetric unit. The (alpha beta)6 particle is dumbbell-shaped, with each (alpha beta)3 unit being approximately spherical, with a diameter of about 65 A. There is a large central cavity approximately 30 A deep around the molecular 3-fold axis of the (alpha beta)3 unit. The 3-fold related active site pockets are located around the bottom of this cavity and are separated from each other by a distance of approximately 23 A. The inner portion of each (alpha beta) unit, which lies near the interface between the two (alpha beta)3 particles, consists mainly of random coil with several small helical and sheet regions. The outer region of each (alpha beta) unit has an unusual structure consisting of two overlapping, predominantly antiparallel beta-pleated sheets, lined on each side by an alpha-helix. The walls of the central cavity are formed by the 3-fold repeat of two strands from this beta-sandwich structure and one of the helices.     

Designed potent multivalent chemoattractants for Escherichia coli.

Bacterial chemotactic responses are initiated when certain small molecules (i.e., carbohydrates, amino acids) interact with bacterial chemoreceptors. Although bacterial chemotaxis has been the subject of intense investigations, few have explored the influence of attractant structure on signal generation and chemotaxis. Previously, we found that polymers bearing multiple copies of galactose interact with the chemoreceptor Trg via the periplasmic binding protein glucose/galactose binding protein (GGBP). These synthetic multivalent ligands were potent agonists of Escherichia coli chemotaxis. Here, we report on the development of a second generation of multivalent attractants that possess increased chemotactic activities. Strikingly, the new ligands can alter bacterial behavior at concentrations 10-fold lower than those required with the original displays; thus, they are some of the most potent synthetic chemoattractants known. The potency depends on the number of galactose moieties attached to the oligomer backbone and the length of the linker tethering these carbohydrates. Our investigations reveal the plasticity of GGBP; it can bind and mediate responses to several carbohydrates and carbohydrate derivatives. These attributes of GGBP may underlie the ability of bacteria to sense a variety of ligands with relatively few receptors. Our results provide insight into the design and development of compounds that can modulate bacterial chemotaxis and pathogenicity.     

Helix swapping between two alpha/beta barrels: crystal structure of phosphoenolpyruvate mutase with bound Mg(2+)-oxalate.

BACKGROUND: Phosphonate compounds are important secondary metabolites in nature and, when linked to macromolecules in eukaryotes, they might play a role in cell signaling. The first obligatory step in the biosynthesis of phosphonates is the formation of a carbon-phosphorus bond by converting phosphoenolpyruvate (PEP) to phosphonopyruvate (P-pyr), a reaction that is catalyzed by PEP mutase. The PEP mutase functions as a tetramer and requires magnesium ions (Mg2+). RESULTS: The crystal structure of PEP mutase from the mollusk Mytilus edulis, bound to the inhibitor Mg(2+)-oxalate, has been determined using multiwavelength anomalous diffraction, exploiting the selenium absorption edge of a selenomethionine-containing protein. The structure has been refined at 1.8 A resolution. PEP mutase adopts a modified alpha/beta barrel fold, in which the eighth alpha helix projects away from the alpha/beta barrel instead of packing against the beta sheet. A tightly associated dimer is formed, such that the two eighth helices are swapped, each packing against the beta sheet of the neighboring molecule. A dimer of dimers further associates into a tetramer. Mg(2+)-oxalate is buried close to the center of the barrel, at the C-terminal ends of the beta strands. CONCLUSIONS: The tetramer observed in the crystal is likely to be physiologically relevant. Because the Mg(2+)-oxalate is inaccessible to solvent, substrate binding and dissociation might be accompanied by conformational changes. A mechanism involving a phosphoenzyme intermediate is proposed, with Asp58 acting as the nucleophilic entity that accepts and delivers the phosphoryl group. The active-site architecture and the chemistry performed by PEP mutase are different from other alpha/beta-barrel proteins that bind pyruvate or PEP, thus the enzyme might represent a new family of alpha/beta-barrel proteins.     

Properties of Mutants in Galactose Taxis and Transport

beta-Methylgalactoside (mgl) permease mutants of Escherichia coli, which are defective in three genes, mglA, mglB, and mglC, were assayed for galactose taxis and galactose transport. The mglB product is the galactose-binding protein. Previous evidence, supported by our new findings, shows that the galactose-binding protein is the recognition component for galactose taxis as well as for galactose transport. Most mutants defective in mglB showed strong effects on both chemotaxis and transport; however, a couple showed effects chiefly on one process or the other, thus allowing a separation of chemotaxis and transport. The mglA and mglC products have not yet been identified, but they must be components of the galactose transport machinery since mutants defective in mglA or mglC, or both, showed strongly reduced transport. Although some of these mutants showed little chemotaxis, most gave close to wild-type chemotactic responses. Thus, transport is not required for galactose taxis. The bacteria detect changes in the fraction of binding protein associated with galactose, not changes in the rate of transport.     

The NMR structure of the sensory domain of the membranous two-component fumarate sensor (histidine protein kinase) DcuS of Escherichia coli

The structure of the water-soluble, periplasmic domain of the fumarate sensor DcuS (DcuS-pd) has been determined by NMR spectroscopy in solution. DcuS is a prototype for a sensory histidine kinase with transmembrane signal transfer. DcuS belongs to the CitA family of sensors that are specific for sensing di- and tricarboxylates. The periplasmic domain is folded autonomously and shows helices at the N and the C terminus, suggesting direct linking or connection to helices in the two transmembrane regions. The structure constitutes a novel fold. The nearest structural neighbor is the Per-Arnt-Sim domain of the photoactive yellow protein that binds small molecules covalently. Residues Arg107, His110, and Arg147 are essential for fumarate sensing and are found clustered together. The structure constitutes the first periplasmic domain of a two component sensory system and is distinctly different from the aspartate sensory domain of the Tar chemotaxis sensor.     

Residues in the alpha helix 7 of the bacterial maltose binding protein which are important in interactions with the Mal FGK2 complex.

The periplasmic maltose binding protein, MalE, is a major element in maltose transport and in chemotaxis towards this sugar. Previous genetic analysis of the MalE protein revealed functional domains involved in transport and chemotactic functions. Among them the surface located alpha helix 7, which is part of the C-lobe, one of the two lobes forming the three dimensional structure of MalE. Small deletions in this region abolished maltose transport, although maintaining wild-type affinity and specificity as well as a normal chemoreceptor function. It was suggested that alpha helix 7 may be implicated in interactions between the maltose binding protein and the membrane-bound protein complex (Duplay P, Szmelcman S. 1987. Silent and functional changes in the periplasmic maltose binding protein of Escherichia coli K12. II. Chemotaxis towards maltose. J Mol Biol 194:675-678: Duplay P, Szmelcman S, Bedouelle H, Hofnung M. 1987. Silent and functional changes in the periplasmic maltose binding protein of Escherichia coli K12. I: Transport of maltose. J Mol Biol 194:663-673). In this study, we submitted a region of 14 residues--Asp 207 to Gly 220--encompassing alpha helix 7, to genetic analysis by oligonucleotide mediated random mutagenesis. Out of 127 identified mutations, twelve single and five double mutants with normal affinities towards maltose were selected for further investigation. Two types of mutations were characterized, silent mutations that did not affect maltose transport and mutations that heavily impaired transport kinetics, even thought the maltose binding capacity of the mutant proteins remained normal. Three substitutions at Tyr 210 (Y210S, Y210L, Y210N) drastically reduced maltose transport. One substitution at Ala 213 (A213I) and one substitution at Glu 214 (E214K) also impaired transport. These three identified residues, Tyr 210, Ala 213, and Glu 214, which are constituents of alpha helix 7, therefore seem to play some important role in maltose transport, most probably in a productive interaction between the MalE protein and the membrane bound MalFGK2 complex.     

The structure of yeast enolase at 2.25-A resolution. An 8-fold beta + alpha-barrel with a novel beta beta alpha alpha (beta alpha)6 topology

The three-dimensional structure of yeast enolase has been determined by the multiple isomorphous replacement method followed by the solvent flattening technique. A polypeptide model, corresponding with the known amino acid sequence, has been fitted to the electron density map. Crystallographic restrained least-squares refinement of the model without solvent gave R = 20.0% for 6-2.25-A resolution with good geometry. A model with 182 water molecules and 1 sulfate which is still being refined has presently R = 17.0%. The molecule is a dimer with subunits related by 2-fold crystallographic symmetry. The subunit has dimensions 60 X 55 X 45 A and is built from two domains. The smaller N-terminal domain has an alpha + beta structure based on a three-stranded antiparallel meander and four helices. The main domain is an 8-fold beta + alpha-barrel. The enolase barrel is, however, different from the triose phosphate isomerase barrel; its topology is beta beta alpha alpha (beta alpha)6 rather than (beta alpha)8 as found in triose phosphate isomerase. The inner beta-barrel is not entirely parallel, the second strand is antiparallel to the other strands, and the direction of the first helix is also reversed with respect to the other helices. This supports the hypothesis that some enzymes evolved independently producing the stable structure of beta alpha barrels with either enolase or triose phosphate isomerase topology. The active site of enolase is located at the carboxylic end of the barrel. A fragment of the N-terminal domain and two long loops protruding from the barrel domain form a wide crevice leading to the active site region. Asp246, Glu295, and Asp320 are the ligands of the conformational cation. Other residues in the active site region are Glu168, Asp321, Lys345, and Lys396.     

FeeM, an N-acyl amino acid synthase from an uncultured soil microbe: structure, mechanism, and acyl carrier protein binding

Attempts to access antibiotics by capturing biosynthetic genes and pathways directly from environmental DNA, which is overwhelmingly derived from uncultured bacteria, have revealed a large and previously unknown family of N-acyl amino acid synthases (NASs). The structure of the NAS FeeM reveals structural similarity to the GCN5-related N-acyl transferases and acylhomoserine lactone synthases. The overall structure has a central beta sheet with alpha helices on both sides. A bound product at a cleft in the beta sheet identifies the active site and the structural basis for catalysis, and sequence conservation in this region indicates a bias for recognition over speed. FeeM interacts with an acyl carrier protein (FeeL), and the structure, mutagenesis, and enzymatic measurements reveal that a small hydrophobic pocket in alpha helix 5 dominates binding of FeeM to FeeL. The structural and mechanistic analyses suggest that the products of FeeM could be bacterial signaling agents.     

The Agrobacterium tumefaciens virulence gene chvE is part of a putative ABC-type sugar transport operon.

The Agrobacterium tumefaciens virulence determinant ChvE is a periplasmic binding protein which participates in chemotaxis and virulence gene induction in response to monosaccharides which occur in the plant wound environment. The region downstream of the A. tumefaciens chvE gene was cloned and sequenced for nucleotide and expression analysis. Three open reading frames transcribed in the same direction as chvE were revealed. The first two, together with chvE, encode putative proteins of a periplasmic binding protein-dependent sugar uptake system, or ABC-type (ATP binding cassette) transporter. The third open reading frame encodes a protein of unknown function. The deduced transporter gene products are related on the amino acid level to bacterial sugar transporters and probably function in glucose and galactose uptake. We have named these genes gguA, -B, and -C, for glucose galactose uptake. Mutations in gguA, gguB, or gguC do not affect virulence of A. tumefaciens on Kalanchoe diagremontiana; growth on 1 mM galactose, glucose, xylose, ribose, arabinose, fucose, or sucrose; or chemotaxis toward glucose, galactose, xylose, or arabinose.     

Three-dimensional structure of rat acid phosphatase.

The crystal structure of recombinant rat prostatic acid phosphatase was determined to 3 A resolution with protein crystallographic methods. The enzyme subunit is built up of two domains, an alpha/beta domain consisting of a seven-stranded mixed beta-sheet with helices on both sides of the sheet and a smaller alpha domain. Two disulfide bridges between residues 129-340 and 315-319 were found. Electron density at two of the glycosylation sites for parts of the carbohydrate moieties was observed. The dimer of acid phosphatase is formed through two-fold interactions of edge strand 3 from one subunit with strand 3 from the second subunit, thus extending the beta-sheet from seven to 14 strands. Other subunit-subunit interactions involve conserved residues from loops between helices and beta-strands. The fold of the alpha/beta domain is similar to the fold observed in phosphoglycerate mutase. The active site is at the carboxy end of the parallel strands of the alpha/beta domain. There is a strong residual electron density at the phosphate binding site which probably represents a bound chloride ion. Biochemical properties and results from site-directed mutagenesis experiments of acid phosphatase are correlated to the three-dimensional structure.     

Crystal structure of MJ1247 protein from M. jannaschii at 2.0 A resolution infers a molecular function of 3-hexulose-6-phosphate isomerase

The crystal structure of the hypothetical protein MJ1247 from Methanococccus jannaschii at 2 A resolution, a detailed sequence analysis, and biochemical assays infer its molecular function to be 3-hexulose-6-phosphate isomerase (PHI). In the dissimilatory ribulose monophosphate (RuMP) cycle, ribulose-5-phosphate is coupled to formaldehyde by the 3-hexulose-6-phosphate synthase (HPS), yielding hexulose-6-phosphate, which is then isomerized to fructose-6-phosphate by the enzyme 3-hexulose-6-phosphate isomerase. MJ1247 is an alpha/beta structure consisting of a five-stranded parallel beta sheet flanked on both sides by alpha helices, forming a three-layered alpha-beta-alpha sandwich. The fold represents the nucleotide binding motif of a flavodoxin type. MJ1247 is a tetramer in the crystal and in solution and each monomer has a folding similar to the isomerase domain of glucosamine-6-phosphate synthase (GlmS).     

The crystal structure of a thermophilic glucose binding protein reveals adaptations that interconvert mono and di-saccharide binding sites

Periplasmic binding proteins (PBPs) comprise a protein superfamily that is involved in prokaryotic solute transport and chemotaxis. These proteins have been used to engineer reagentless biosensors to detect natural or non-natural ligands. There is considerable interest in obtaining very stable members of this superfamily from thermophilic bacteria to use as robust engineerable parts in biosensor development. Analysis of the recently determined genome sequence of Thermus thermophilus revealed the presence of more than 30 putative PBPs in this thermophile. One of these is annotated as a glucose binding protein (GBP) based on its genetic linkage to genes that are homologous to an ATP-binding cassette glucose transport system, although the PBP sequence is homologous to periplasmic maltose binding proteins (MBPs). Here we present the cloning, over-expression, characterization of cognate ligands, and determination of the X-ray crystal structure of this gene product. We find that it is a very stable (apo-protein Tm value is 100(+/- 2) degrees C; complexes 106(+/- 3) degrees C and 111(+/- 1) degrees C for glucose and galactose, respectively) glucose (Kd value is 0.08(+/- 0.03) microM) and galactose (Kd value is 0.94(+/- 0.04) microM) binding protein. Determination of the X-ray crystal structure revealed that this T. thermophilus glucose binding protein (ttGBP) is structurally homologous to MBPs rather than other GBPs. The di or tri-saccharide ligands in MBPs are accommodated in long relatively shallow grooves. In the ttGBP binding site, this groove is partially filled by two loops and an alpha-helix, which create a buried binding site that allows binding of only monosaccharides. Comparison of ttGBP and MBP provides a clear example of structural adaptations by which the size of ligand binding sites can be controlled in the PBP super family.     

Dihydrofolate reductase from Lactobacillus casei. Stereochemistry of NADPH binding.

The NADPH molecule binds to dihydrofolate reductase in an extended conformation. Several of the individual dihedral angles, especially in the adenine mononucleotide portion of the coenzyme, differ from their minimum energy conformations. The ribose phosphate portions of the coenzyme are involved in numerous specific hydrogen-bonded and charge-charge interactions. The adenine ring resides in an apparently nonspecific hydrophobic cleft and the nicotinamide ring is bound within an intricately constructed cavity, one wall of which includes the pyrazine ring of bound methotrexate. Two rather extended loops (residues 10 to 24 and 117 to 135) connecting beta A to alpha B and beta F to beta G, respectively, move 2 to 3 A when NADPH binds to dihydrofolate reductase. No overall structural homology is evident between the dinucleotide binding domains of dihydrofolate reductase on the one hand and the four NAD+-dependent dehydrogenases of known structure on the other. However, binding does occur in both cases at the carboxyl edge of a region of parallel beta sheet flanked by a pair of alpha helices.     

Family of G protein alpha chains: amphipathic analysis and predicted structure of functional domains

The G proteins transduce hormonal and other signals into regulation of enzymes such as adenylyl cyclase and retinal cGMP phosphodiesterase. Each G protein contains an alpha subunit that binds and hydrolyzes guanine nucleotides and interacts with beta gamma subunits and specific receptor and effector proteins. Amphipathic and secondary structure analysis of the primary sequences of five different alpha chains (bovine alpha s, alpha t1 and alpha t2, mouse alpha i, and rat alpha o) predicted the secondary structure of a composite alpha chain (alpha avg). The alpha chains contain four short regions of sequence homologous to regions in the GDP binding domain of bacterial elongation factor Tu (EF-Tu). Similarities between the predicted secondary structures of these regions in alpha avg and the known secondary structure of EF-Tu allowed us to construct a three-dimensional model of the GDP binding domain of alpha avg. Identification of the GDP binding domain of alpha avg defined three additional domains in the composite polypeptide. The first includes the amino terminal 41 residues of alpha avg, with a predicted amphipathic alpha helical structure; this domain may control binding of the alpha chains to the beta gamma complex. The second domain, containing predicted beta strands and alpha helices, several of which are strongly amphipathic, probably contains sequences responsible for interaction of alpha chains with effector enzymes. The predicted structure of the third domain, containing the carboxy terminal 100 amino acids, is predominantly beta sheet with an amphipathic alpha helix at the carboxy terminus. We propose that this domain is responsible for receptor binding.(ABSTRACT TRUNCATED AT 250 WORDS)