Structure of the D-alanylgriseoluteic acid biosynthetic protein EhpF, an atypical member of the ANL superfamily of adenylating enzymes.

The structure of EhpF, a 41 kDa protein that functions in the biosynthetic pathway leading to the broad-spectrum antimicrobial compound D-alanylgriseoluteic acid (AGA), is reported. A cluster of approximately 16 genes, including ehpF, located on a 200 kbp plasmid native to certain strains of Pantoea agglomerans encodes the proteins that are required for the conversion of chorismic acid to AGA. Phenazine-1,6-dicarboxylate has been identified as an intermediate in AGA biosynthesis and deletion of ehpF results in accumulation of this compound in vivo.

Probing anomalous structural features in polypurine tract-containing RNA-DNA hybrids with neomycin B.

During (-)-strand DNA synthesis in retroviruses and Saccharomyces cerevisiae LTR retrotransposons, a purine rich region of the RNA template, known as the polypurine tract (PPT), is resistant to RNase H-mediated hydrolysis and subsequently serves as a primer for (+)-strand, DNA-dependent DNA synthesis. Although HIV-1 and Ty3 PPT sequences share no sequence similarity beyond the fact that both include runs of purine ribonucleotides, it has been suggested that these PPTs are processed by their cognate reverse transcriptases (RTs) through a common molecular mechanism. Here, we have used the aminoglycoside neomycin B (NB) to examine which structural features of the Ty3 PPT contribute to specific recognition and processing by its cognate RT.

The Methanothermobacter thermautotrophicus MCM helicase is active as a hexameric ring.

The minichromosome maintenance (MCM) complex is thought to function as the replicative helicase in archaea and eukarya. The structure of the single MCM protein homologue from the archaeon Methanothermobacter thermautotrophicus is not yet clear, and hexameric, heptameric, octameric, and dodecameric structures, open rings, and filamentous structures have been reported. Using a combination of biochemical and structural analysis, it is shown here that the M.

Crystallization and X-ray diffraction analysis of salicylate synthase, a chorismate-utilizing enyme involved in siderophore biosynthesis.

Bacteria have evolved elaborate schemes that help them thrive in environments where free iron is severely limited. Siderophores such as yersiniabactin are small iron-scavenging molecules that are deployed by bacteria during iron starvation. Several studies have linked siderophore production and virulence.

MitoMorphy: an alignment and annotation tool for human mitochondrial DNA polymorphisms.

MitoMorphy uses a number of publicly available human mitochondrial DNA (mtDNA) sequences from different ethnic groups to compare and annotate the associated polymorphic data. It provides an integrated display of mtDNA sequence comparison, sequence variation, and annotation for 695 different mtDNA sequences from many different ethnic groups around the world.

Current concepts of active defense in plants.

A growing body of evidence indicates that elicitation of primary active defense responses results from a recognition event frequently involving protein-protein interactions. Most pathogen avirulence determinants eliciting resistance gene-dependent responses have been shown to be proteins with no apparent enzymic activity. Disruption of the tertiary and quaternary structure of these proteins abolishes their elicitor activity.

Phototransduction: crystal clear.

Vertebrate visual phototransduction represents one of the best-characterized G-protein-coupled receptor-mediated signaling pathways. Structural analyses of rhodopsin, G protein, arrestin and several other phototransduction components have revealed common folds and motifs that are important for function. Static and dynamic information has been acquired through the application of X-ray diffraction, solution and solid-state nuclear magnetic resonance spectroscopy's, electron and atomic force microscopy's, and a host of indirect structural methods.

Crystal structure of the YajQ protein from Haemophilus influenzae reveals a tandem of RNP-like domains.

A hypothetical protein encoded by the gene YajQ of Haemophilus influenzae was selected, as part of a structural genomics project, for X-ray crystallographic structure determination and analysis to assist with the functional assignment. The protein is present in most bacteria, but not in archaea or eukaryotes. The amino acid sequence has no homology to that of other proteins.

JE-TROSY: combined J- and TROSY-spectroscopy for the measurement of one-bond couplings in macromolecules.

With the application of RDCs in high-resolution NMR studies of macromolecules, there has been an interest in the development of accurate, sensitive methods for measuring 15N-1H and 13C-1H one-bond coupling constants. Most methods for determining these couplings are based on the measurement of the displacement between cross-peak components in J-coupled spectra. However, for large macromolecules and macromolecular complexes, these methods are often unreliable since differential relaxation can significantly broaden one of the multiplet components (i.

Proflavine acts as a Rev inhibitor by targeting the high-affinity Rev binding site of the Rev responsive element of HIV-1.

Rev is an essential regulatory HIV-1 protein that binds the Rev responsive element (RRE) within the env gene of the HIV-1 RNA genome, activating the switch between viral latency and active viral replication. Previously, we have shown that selective incorporation of the fluorescent probe 2-aminopurine (2-AP) into a truncated form of the RRE sequence (RRE-IIB) allowed the binding of an arginine-rich peptide derived from Rev and aminoglycosides to be characterized directly by fluorescence methods. Using these fluorescence and nuclear magnetic resonance (NMR) methods, proflavine has been identified, through a limited screen of selected small heterocyclic compounds, as a specific and high-affinity RRE-IIB binder which inhibits the interaction of the Rev peptide with RRE-IIB.

Increased Sensitivity of Fluorescence Detection Using Metallic Nanoparticles.

A new technique called radiative decay engineering can be used to modify fluorescence emissions by changing the free space conditions around the fluorophores.

Assisting functional assignment for hypothetical Heamophilus influenzae gene products through structural genomics.

The three-dimensional structures of Haemophilus influenzae proteins whose biological functions are unknown are being determined as part of a structural genomics project to ask whether structural information can assist in assigning the functions of proteins. The structures of the hypothetical proteins are being used to guide further studies and narrow the field of such studies for ultimately determining protein function. An outline of the structural genomics methodological approach is provided along with summaries of a number of completed and in progress crystallographic and NMR structure determinations.

Observation of H-bond mediated 3hJH2H3 coupling constants across Watson-Crick AU base pairs in RNA.

3hJH2H3 trans-hydrogen bond scalar coupling constants have been observed for the first time in Watson-Crick AU base pairs in uniformly 15N-labeled RNA oligonucleotides using a new 2hJNN-HNN-E. COSY experiment. The experiment utilizes adenosine H2 (AH2) for original polarization and detection, while employing 2hJNN couplings for coherence transfer across the hydrogen bonds (H-bonds).

Algal rhodopsins: phototaxis receptors found at last.

The discovery of two distinct Chlamydomonas sensory receptors responsible for phototaxis reveals additional diversity among the microbial rhodopsins. Sequence and architecture comparisons among this growing family highlight key components for light-responsive functions.

Crystal structure of the YjeE protein from Haemophilus influenzae: a putative Atpase involved in cell wall synthesis.

A hypothetical protein encoded by the gene YjeE of Haemophilus influenzae was selected as part of a structural genomics project for X-ray analysis to assist with the functional assignment. The protein is considered essential to bacteria because the gene is present in virtually all bacterial genomes but not in those of archaea or eukaryotes. The amino acid sequence shows no homology to other proteins except for the presence of the Walker A motif G-X-X-X-X-G-K-T that indicates the possibility of a nucleotide-binding protein.

The Biological Macromolecule Crystallization Database: crystallization procedures and strategies.

The Biological Macromolecule Crystallization Database (BMCD) archives crystallization data from published reports for all forms of biological macromolecules that have produced crystals suitable for X-ray diffraction studies. The information includes the crystallization conditions, crystal data, comments about the crystallization procedure and information on the biological macromolecule or biological macromolecule complex. Crystallization procedures, including fast screens and more general procedures, can be developed effectively using this web-based resource (http://wwwbmcd.

Grafting segments from the extracellular surface of CCR5 onto a bacteriorhodopsin transmembrane scaffold confers HIV-1 coreceptor activity.

Components from the extracellular surface of CCR5 interact with certain macrophage-tropic strains of human immunodeficiency virus type 1 (HIV-1) to mediate viral fusion and entry. To mimic these viral interacting site(s), the amino-terminal and extracellular loop segments of CCR5 were linked in tandem to form concatenated polypeptides, or grafted onto a seven-transmembrane bacteriorhodopsin scaffold to generate several chimeras. The chimera studies identified specific regions in CCR5 that confer HIV-1 coreceptor function, structural rearrangements in the transmembrane region that may modulate this activity, and a role for the extracellular surface in folding and assembly.

Crystal structure of dephospho-coenzyme A kinase from Haemophilus influenzae.

Dephospho-coenzyme A kinase catalyzes the final step in CoA biosynthesis, the phosphorylation of the 3'-hydroxyl group of ribose using ATP as a phosphate donor. The protein from Haemophilus influenzae was cloned and expressed, and its crystal structure was determined at 2.0-A resolution in complex with ATP.

The structure of the T127L/S128A mutant of cAMP receptor protein facilitates promoter site binding.

The x-ray crystal structure of the cAMP-ligated T127L/S128A double mutant of cAMP receptor protein (CRP) was determined to a resolution of 2.2 A. Although this structure is close to that of the x-ray crystal structure of cAMP-ligated CRP with one subunit in the open form and one subunit in the closed form, a bound syn-cAMP is clearly observed in the closed subunit in a third binding site in the C-terminal domain.

Macromolecular Crystallography and Structural Biology Databases at NIST.

In the late 1970s, macromolecular crystallography at NIST began with collaboration between NIST and NIH to establish a single-crystal neutron diffractometer. This instrument was constructed and employed to solve a number of crystal structures: bovine ribonuclease A, bovine-ribonuclease-uridine vanadate complex, and porcine insulin. In the mid 1980s a Biomolecular Structure Group was created establishing NIST capabilities in biomolecular singe-crystal x-ray diffraction.

Escherichia coli uracil DNA glycosylase: NMR characterization of the short hydrogen bond from His187 to uracil O2.

Uracil DNA glycosylase (UDG) cleaves the glycosidic bond of deoxyuridine in DNA using a hydrolytic mechanism, with an overall catalytic rate enhancement of 10(12)-fold over the solution reaction. The nature of the enzyme-substrate interactions that lead to this large rate enhancement are key to understanding enzymatic DNA repair. Using (1)H and heteronuclear NMR spectroscopy, we have characterized one such interaction in the ternary product complex of Escherichia coli UDG, the short (2.

Functionally discrete mimics of light-activated rhodopsin identified through expression of soluble cytoplasmic domains.

Numerous studies on the seven-helix receptor rhodopsin have implicated the cytoplasmic loops and carboxyl-terminal region in the binding and activation of proteins involved in visual transduction and desensitization. In our continuing studies on rhodopsin folding, assembly, and structure, we have attempted to reconstruct the interacting surface(s) for these proteins by inserting fragments corresponding to the cytoplasmic loops and/or the carboxyl-terminal tail of bovine opsin either singly, or in combination, onto a surface loop in thioredoxin. The purpose of the thioredoxin fusion is to provide a soluble scaffold for the cytoplasmic fragments thereby allowing them sufficient conformational freedom to fold to a structure that mimics the protein-binding sites on light-activated rhodopsin.

The 1.30 A resolution structure of the Bacillus subtilis chorismate mutase catalytic homotrimer.

The crystal structure of the Bacillus subtilis chorismate mutase, an enzyme of the aromatic amino acids biosynthetic pathway, was determined to 1.30 A resolution. The structure of the homotrimer was determined by molecular replacement using orthorhombic crystals of space group P2(1)2(1)2(1) with unit-cell parameters a = 52.

Folding and assembly in rhodopsin. Effect of mutations in the sixth transmembrane helix on the conformation of the third cytoplasmic loop.

Previous studies on bovine opsin folding and assembly have identified an amino-terminal fragment, EF(1-232), which folds and inserts into a membrane only after coexpression with its complementary carboxyl-terminal fragment, EF(233-348). To further characterize this interaction, EF(1-232) production was examined upon coexpression with carboxyl-terminal fragments of varying length and/or amino acid composition. These included fragments with incremental deletions of the third cytoplasmic loop (TH(241-348) and EF(249-348)), a fragment composed of the third cytoplasmic loop and sixth transmembrane helix (HF(233-280)), a fragment composed of the sixth and seventh transmembrane helices (FG(249-312)), and EF(233-348) and TH(241-348) fragments with Pro-267 or Trp-265 mutations.

The three-dimensional structures of two isoforms of nucleoside diphosphate kinase from bovine retina.

The crystal structures of two isoforms of nucleoside diphosphate kinase from bovine retina overexpressed in Escherischia coli have been determined to 2.4 A resolution. Both the isoforms, NBR-A and NBR-B, are hexameric and the fold of the monomer is in agreement with NDP-kinase structures from other biological sources.