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Aled Edwards

Publications and source records attributed to Aled Edwards.

16 recordsLinked to original sources
PubMed2006

Methyltransferase that modifies guanine 966 of the 16 S rRNA: functional identification and tertiary structure.

N(2)-Methylguanine 966 is located in the loop of Escherichia coli 16 S rRNA helix 31, forming a part of the P-site tRNA-binding pocket. We found yhhF to be a gene encoding for m(2)G966 specific 16 S rRNA methyltransferase. Disruption of the yhhF gene by kanamycin resistance marker leads to a loss of modification at G966. The modification could be rescued by expression of recombinant protein from the plasmid carrying the yhhF gene. Moreover, purified m(2)G966 methyltransferase, in the presence of S-adenosylomethionine (AdoMet), is able to methylate 30 S ribosomal subunits that were purified from yhhF knock-out strain in vitro. The methylation is specific for G966 base of the 16 S rRNA. The m(2)G966 methyltransferase was crystallized, and its structure has been determined and refined to 2.05A(.) The structure closely resembles RsmC rRNA methyltransferase, specific for m(2)G1207 of the 16 S rRNA. Structural comparisons and analysis of the enzyme active site suggest modes for binding AdoMet and rRNA to m(2)G966 methyltransferase. Based on the experimental data and current nomenclature the protein expressed from the yhhF gene was renamed to RsmD. A model for interaction of RsmD with ribosome has been proposed.

Escherichia coli↗
PubMed2006

Genome-scale protein expression and structural biology of Plasmodium falciparum and related Apicomplexan organisms.

Parasites from the protozoan phylum Apicomplexa are responsible for diseases, such as malaria, toxoplasmosis and cryptosporidiosis, all of which have significantly higher rates of mortality and morbidity in economically underdeveloped regions of the world. Advances in vaccine development and drug discovery are urgently needed to control these diseases and can be facilitated by production of purified recombinant proteins from Apicomplexan genomes and determination of their 3D structures. To date, both heterologous expression and crystallization of Apicomplexan proteins have seen only limited success. In an effort to explore the effectiveness of producing and crystallizing proteins on a genome-scale using a standardized methodology, over 400 distinct Plasmodium falciparum target genes were chosen representing different cellular classes, along with select orthologues from four other Plasmodium species as well as Cryptosporidium parvum and Toxoplasma gondii. From a total of 1008 genes from the seven genomes, 304 (30.2%) produced purified soluble proteins and 97 (9.6%) crystallized, culminating in 36 crystal structures. These results demonstrate that, contrary to previous findings, a standardized platform using Escherichia coli can be effective for genome-scale production and crystallography of Apicomplexan proteins. Predictably, orthologous proteins from different Apicomplexan genomes behaved differently in expression, purification and crystallization, although the overall success rates of Plasmodium orthologues do not differ significantly. Their differences were effectively exploited to elevate the overall productivity to levels comparable to the most successful ongoing structural genomics projects: 229 of the 468 target genes produced purified soluble protein from one or more organisms, with 80 and 32 of the purified targets, respectively, leading to crystals and ultimately structures from one or more orthologues.

Animals↗
PubMed2006

Structure of SAICAR synthase from Thermotoga maritima at 2.2 angstroms reveals an unusual covalent dimer.

As a part of a structural genomics program, the 2.2 angstroms resolution crystal structure of the PurC gene product from Thermotoga maritima has been solved. This 26.2 kDa protein belongs to the phophoribosylaminoimidazole-succinocarboxamide or SAICAR synthase family of enzymes, the members of which are involved in de novo purine biosynthesis. SAICAR synthase can be divided into three subdomains: two alpha+beta regions exhibiting structural homology with ATP-binding proteins and a carboxy-terminal subdomain of two alpha-helices. The asymmetric unit contains two copies of the protein which are covalently linked by a disulfide bond between Cys126(A) and Cys126(B). This 230-amino-acid protein exhibits high structural homology with SAICAR synthase from baker's yeast. The protein structure is described and compared with that of the ATP-SAICAR synthase complex from yeast.

Amino Acid Sequence↗
PubMed2005

The Drosophila nuclear receptor e75 contains heme and is gas responsive.

Nuclear receptors are a family of transcription factors with structurally conserved ligand binding domains that regulate their activity. Despite intensive efforts to identify ligands, most nuclear receptors are still "orphans." Here, we demonstrate that the ligand binding pocket of the Drosophila nuclear receptor E75 contains a heme prosthetic group. E75 absorption spectra, resistance to denaturants, and effects of site-directed mutagenesis indicate a single, coordinately bound heme molecule. A correlation between the levels of E75 expression and the levels of available heme suggest a possible role as a heme sensor. The oxidation state of the heme iron also determines whether E75 can interact with its heterodimer partner DHR3, suggesting an additional role as a redox sensor. Further, the E75-DHR3 interaction is also regulated by the binding of NO or CO to the heme center, suggesting that E75 may also function as a diatomic gas sensor. Possible mechanisms and roles for these interactions are discussed.

Amino Acid Sequence↗
PubMed2005

A high throughput screen to identify substrates for the ubiquitin ligase Rsp5.

Ubiquitin-protein ligases (E3s) are implicated in various human disorders and are attractive targets for therapeutic intervention. Although most cellular proteins are ubiquitinated, ubiquitination cannot be linked directly to a specific E3 for a large fraction of these proteins, and the substrates of most E3 enzymes are unknown. We have developed a luminescent assay to detect ubiquitination in vitro, which is more quantitative, effective, and sensitive than conventional ubiquitination assays. By taking advantage of the abundance of purified proteins made available by genomic efforts, we screened hundreds of purified yeast proteins for ubiquitination, and we identified previously reported and novel substrates of the yeast E3 ligase Rsp5. The relevance of these substrates was confirmed in vivo by showing that a number of them interact genetically with Rsp5, and some were ubiquitinated by Rsp5 in vivo. The combination of this sensitive assay and the availability of purified substrates will enable the identification of substrates for any purified E3 enzyme.

Endosomal Sorting Complexes Required for Transport↗
PubMed2004

The crystal structure of Escherichia coli MoaB suggests a probable role in molybdenum cofactor synthesis.

The crystal structure of Escherichia coli MoaB was determined by multiwavelength anomalous diffraction phasing and refined at 1.6-A resolution. The molecule displayed a modified Rossman fold. MoaB is assembled into a hexamer composed of two trimers. The monomers have high structural similarity with two proteins, MogA and MoeA, from the molybdenum cofactor synthesis pathway in E. coli, as well as with domains of mammalian gephyrin and plant Cnx1, which are also involved in molybdopterin synthesis. Structural comparison between these proteins and the amino acid conservation patterns revealed a putative active site in MoaB. The structural analysis of this site allowed to advance several hypothesis that can be tested in further studies.

Arabidopsis Proteins↗
PubMed2003

High-throughput protein crystallization.

High-throughput structural biology is a focus of a number of academic and pharmaceutical laboratories around the world. The use of X-ray crystallography in these efforts is critically dependent on high-throughput protein crystallization. The application of current protocols yields crystal leads for approximately 30% of the input proteins and well-diffracting crystals for a smaller fraction. Increasing the success rate will require a multidisciplinary approach that must invoke techniques from molecular biology, protein biochemistry, biophysics, artificial intelligence, and automation.

Automation↗
PubMed2003

Crystal complexes of a predicted S-adenosylmethionine-dependent methyltransferase reveal a typical AdoMet binding domain and a substrate recognition domain.

S-adenosyl-L-methionine-dependent methyltransferases (MTs) are abundant, and highly conserved across phylogeny. These enzymes use the cofactor AdoMet to methylate a wide variety of molecular targets, thereby modulating important cellular and metabolic activities. Thermotoga maritima protein 0872 (TM0872) belongs to a large sequence family of predicted MTs, ranging phylogenetically from relatively simple bacteria to humans. The genes for many of the bacterial homologs are located within operons involved in cell wall synthesis and cell division. Despite preliminary biochemical studies in E. coli and B. subtilis, the substrate specificity of this group of more than 150 proteins is unknown. As part of the Midwest Center for Structural Genomics initiative (www.mcsg.anl.gov), we have determined the structure of TM0872 in complexes with AdoMet and with S-adenosyl-L-homocysteine (AdoHcy). As predicted, TM0872 has a typical MT domain, and binds endogenous AdoMet, or co-crystallized AdoHcy, in a manner consistent with other known MT structures. In addition, TM0872 has a second domain that is novel among MTs in both its location in the sequence and its structure. The second domain likely acts in substrate recognition and binding, and there is a potential substrate-binding cleft spanning the two domains. This long and narrow cleft is lined with positively charged residues which are located opposite the S(+)-CH(3) bond, suggesting that a negatively charged molecule might be targeted for catalysis. However, AdoMet and AdoHcy are both buried, and access to the methyl group would presumably require structural rearrangement. These TM0872 crystal structures offer the first structural glimpses at this phylogenetically conserved sequence family.

Amino Acid Sequence↗
PubMed2003

Solution structure of ribosomal protein S28E from Methanobacterium thermoautotrophicum.

The ribosomal protein S28E from the archaeon Methanobacterium thermoautotrophicum is a component of the 30S ribosomal subunit. Sequence homologs of S28E are found only in archaea and eukaryotes. Here we report the three-dimensional solution structure of S28E by NMR spectroscopy. S28E contains a globular region and a long C-terminal tail protruding from the core. The globular region consists of four antiparallel beta-strands that are arranged in a Greek-key topology. Unique features of S28E include an extended loop L2-3 that folds back onto the protein and a 12-residue charged C-terminal tail with no regular secondary structure and greater flexibility relative to the rest of the protein. The structural and surface resemblance to OB-fold family of proteins and the presence of highly conserved basic residues suggest that S28E may bind to RNA. A broad positively charged surface extending over one side of the beta-barrel and into the flexible C terminus may present a putative binding site for RNA.

Amino Acid Sequence↗
PubMed2002

Aspartate dehydrogenase, a novel enzyme identified from structural and functional studies of TM1643.

The open reading frame TM1643 of Thermotoga maritima belongs to a large family of proteins, with homologues in bacteria, archaea, and eukaryotes. TM1643 is found in an operon with two other genes that encode enzymes involved in the biosynthesis of NAD. In several bacteria, the gene in the position occupied by TM1643 encodes an aspartate oxidase (NadB), which synthesizes iminoaspartate as a substrate for NadA, the next enzyme in the pathway. The amino acid sequence of TM1643 does not share any recognizable homology with aspartate oxidase or with other proteins of known functions or structures. To help define the biological functions of TM1643, we determined its crystal structure at 2.6A resolution and performed a series of screens for enzymatic function. The structure reveals the presence of an N-terminal Rossmann fold domain with a bound NAD(+) cofactor and a C-terminal alpha+beta domain. The structural information suggests that TM1643 may be a dehydrogenase and the active site of the enzyme is located at the interface between the two domains. The enzymatic characterization of TM1643 revealed that it possesses NAD or NADP-dependent dehydrogenase activity toward l-aspartate but no aspartate oxidase activity. The product of the aspartate dehydrogenase activity is also iminoaspartate. Therefore, our studies demonstrate that two different enzymes, an oxidase and a dehydrogenase, may have evolved to catalyze the first step of NAD biosynthesis in prokaryotes. TM1643 establishes a new class of amino acid dehydrogenases.

Amino Acid Oxidoreductases↗
PubMed2002

Crystal structure of Thermotoga maritima 0065, a member of the IclR transcriptional factor family.

Members of the IclR family of transcription regulators modulate signal-dependent expression of genes involved in carbon metabolism in bacteria and archaea. The Thermotoga maritima TM0065 gene codes for a protein (TM-IclR) that is homologous to the IclR family. We have determined the crystal structure of TM-IclR at 2.2 A resolution using MAD phasing and synchrotron radiation. The protein is composed of two domains: the N-terminal DNA-binding domain contains the winged helix-turn-helix motif, and the C-terminal presumed regulatory domain is involved in binding signal molecule. In a proposed signal-binding site, a bound Zn(2+) ion was found. In the crystal, TM-IclR forms a dimer through interactions between DNA-binding domains. In the dimer, the DNA-binding domains are 2-fold related, but the dimer is asymmetric with respect to the orientation of signal-binding domains. Crystal packing analysis showed that TM-IclR dimers form a tetramer through interactions exclusively by signal-binding domains. A model is proposed for binding of IclR-like factors to DNA, and it suggests that signal-dependent transcription regulation is accomplished by affecting an oligomerization state of IclR and therefore its affinity for DNA target.

Amino Acid Sequence↗
PubMed2002

The crystal structure of spermidine synthase with a multisubstrate adduct inhibitor.

Polyamines are essential in all branches of life. Spermidine synthase (putrescine aminopropyltransferase, PAPT) catalyzes the biosynthesis of spermidine, a ubiquitous polyamine. The crystal structure of the PAPT from Thermotoga maritima (TmPAPT) has been solved to 1.5 A resolution in the presence and absence of AdoDATO (S-adenosyl-1,8-diamino-3-thiooctane), a compound containing both substrate and product moieties. This, the first structure of an aminopropyltransferase, reveals deep cavities for binding substrate and cofactor, and a loop that envelops the active site. The AdoDATO binding site is lined with residues conserved in PAPT enzymes from bacteria to humans, suggesting a universal catalytic mechanism. Other conserved residues act sterically to provide a structural basis for polyamine specificity. The enzyme is tetrameric; each monomer consists of a C-terminal domain with a Rossmann-like fold and an N-terminal beta-stranded domain. The tetramer is assembled using a novel barrel-type oligomerization motif.

Adenosine↗
PubMedDate not supplied

Target selection and determination of function in structural genomics.

The first crucial step in any structural genomics project is the selection and prioritization of target proteins for structure determination. There may be a number of selection criteria to be satisfied, including that the proteins have novel folds, that they be representatives of large families for which no structure is known, and so on. The better the selection at this stage, the greater is the value of the structures obtained at the end of the experimental process. This value can be further enhanced once the protein structures have been solved if the functions of the given proteins can also be determined. Here we describe the methods used at either end of the experimental process: firstly, sensitive sequence comparison techniques for selecting a high-quality list of target proteins, and secondly the various computational methods that can be applied to the eventual 3D structures to determine the most likely biochemical function of the proteins in question.

Amino Acid Motifs↗