Scott Cameron

ELM server: a new resource for investigating short functional sites in modular eukaryotic proteins

Multidomain proteins predominate in eukaryotic proteomes. Individual functions assigned to different sequence segments combine to create a complex function for the whole protein. While on-line resources are available for revealing globular domains in sequences, there has hitherto been no comprehensive collection of small functional sites/motifs comparable to the globular domain resources, yet these are as important for the function of multidomain proteins. Short linear peptide motifs are used for cell compartment targeting, protein-protein interaction, regulation by phosphorylation, acetylation, glycosylation and a host of other post-translational modifications. ELM, the Eukaryotic Linear Motif server at http://elm.eu.org/, is a new bioinformatics resource for investigating candidate short non-globular functional motifs in eukaryotic proteins, aiming to fill the void in bioinformatics tools. Sequence comparisons with short motifs are difficult to evaluate because the usual significance assessments are inappropriate. Therefore the server is implemented with several logical filters to eliminate false positives. Current filters are for cell compartment, globular domain clash and taxonomic range. In favourable cases, the filters can reduce the number of retained matches by an order of magnitude or more.

Phospho.ELM: a database of experimentally verified phosphorylation sites in eukaryotic proteins.

BackgroundPost-translational phosphorylation is one of the most common protein modifications. Phosphoserine, threonine and tyrosine residues play critical roles in the regulation of many cellular processes. The fast growing number of research reports on protein phosphorylation points to a general need for an accurate database dedicated to phosphorylation to provide easily retrievable information on phosphoproteins.DescriptionPhospho.ELM http://phospho.elm.eu.org is a new resource containing experimentally verified phosphorylation sites manually curated from the literature and is developed as part of the ELM (Eukaryotic Linear Motif) resource. Phospho.ELM constitutes the largest searchable collection of phosphorylation sites available to the research community. The Phospho.ELM entries store information about substrate proteins with the exact positions of residues known to be phosphorylated by cellular kinases. Additional annotation includes literature references, subcellular compartment, tissue distribution, and information about the signaling pathways involved as well as links to the molecular interaction database MINT. Phospho.ELM version 2.0 contains 1703 phosphorylation site instances for 556 phosphorylated proteins.ConclusionPhospho.ELM will be a valuable tool both for molecular biologists working on protein phosphorylation sites and for bioinformaticians developing computational predictions on the specificity of phosphorylation reactions.

TarO: a target optimisation system for structural biology

TarO (http://www.compbio.dundee.ac.uk/taro) offers a single point of reference for key bioinformatics analyses relevant to selecting proteins or domains for study by structural biology techniques. The protein sequence is analysed by 17 algorithms and compared to 8 databases. TarO gathers putative homologues, including orthologues, and then obtains predictions of properties for these sequences including crystallisation propensity, protein disorder and post-translational modifications. Analyses are run on a high-performance computing cluster, the results integrated, stored in a database and accessed through a web-based user interface. Output is in tabulated format and in the form of an annotated multiple sequence alignment (MSA) that may be edited interactively in the program Jalview. TarO also simplifies the gathering of additional annotations via the Distributed Annotation System, both from the MSA in Jalview and through links to Dasty2. Routes to other information gateways are included, for example to relevant pages from UniProt, COG and the Conserved Domains Database. Open access to TarO is available from a guest account with private accounts for academic use available on request. Future development of TarO will include further analysis steps and integration with the Protein Information Management System (PIMS), a sister project in the BBSRC ‘Structural Proteomics of Rational Targets’ initiative

Comparison of a high-throughput high-content intracellular Leishmania donovani assay with an axenic amastigote assay.

ABSTRACT Visceral leishmaniasis is a neglected tropical disease with significant health impact. The current treatments are poor, and there is an urgent need to develop new drugs. Primary screening assays used for drug discovery campaigns have typically used free-living forms of the Leishmania parasite to allow for high-throughput screening. Such screens do not necessarily reflect the physiological situation, as the disease-causing stage of the parasite resides inside human host cells. Assessing the drug sensitivity of intracellular parasites on scale has recently become feasible with the advent of high-content screening methods. We describe here a 384-well microscopy-based intramacrophage Leishmania donovani assay and compare it to an axenic amastigote system. A panel of eight reference compounds was tested in both systems, as well as a human counterscreen cell line, and our findings show that for most clinically used compounds both axenic and intramacrophage assays report very similar results. A set of 15,659 diverse compounds was also screened using both systems. This resulted in the identification of seven new antileishmanial compounds and revealed a high false-positive rate for the axenic assay. We conclude that the intramacrophage assay is more suited as a primary hit-discovery platform than the current form of axenic assay, and we discuss how modifications to the axenic assay may render it more suitable for hit-discovery.

Specificity and Mechanism of Acinetobacter baumanii Nicotinamidase: Implications for Activation of the Front‐Line Tuberculosis Drug Pyrazinamide

Nicotinamidase (EC 3.5.1.19) catalyzes hydrolysis of nicotinamide to nicotinic acid and ammonia, an important reaction in the NAD salvage pathway. This activity has a fortuitous medical benefit since the Mycobacterium tuberculosis enzyme converts the nicotinamide analogue prodrug pyrazinamide into the bacteriostatic pyrazinoic acid, hence the alternative name, pyrazinamidase (PncA). Pyrazinoic acid inhibits M. tuberculosis type I fatty acid synthase, represses mycolic acid biosynthesis, and appears to affect membrane energetics and acidification of the cytoplasm. It is active against semidormant tubercle bacilli and with rifampicin and isoniazid, forms the front-line tuberculosis treatment. 3] Though studies of PncA have revealed aspects of its structure and biochemical activity there are no structural data on how the enzyme binds and processes physiological ligands. Highresolution crystal structures of Acinetobacter baumanii PncA (AbPncA) complexed with nicotinic acid and pyrazinoic acid now provide direct evidence for the interactions that govern the specificity and mechanism, and of how a valued antibacterial agent is activated. Recombinant AbPncA was prepared, the dimeric, colorless enzyme was purified in high yield and its kinetic properties determined. With pyrazinamide as the substrate the following values were obtained; KM = 106.9 mm, Vmax = 62.8 nmol min , kcat. = 3.1 min , specific activity 132 mmmin 1 mg . These values are comparable to literature values, for example, the specific activity of M. tuberculosis PncA (MtPncA) with pyrazinamide is 82 mmmin 1 mg . Two crystal forms (I and II) were obtained with nicotinic and pyrazinoic acid, respectively, and the structures determined. PncA is a divalent cation-dependent enzyme and activity has been reported with Fe, Mn, and Zn ions. As expected, metal ions were observed in the structures. Inductively coupled plasma-atomic emission spectrometry (ICP-OES) identified that recombinant AbPncA contained Fe and Zn ions in an approximate 1:1 ratio with a trace of Mn present. However, anomalous dispersion measurements are consistent with a higher occupancy of Zn at the active site and the crystallographic models contain that cation. We refer to Zn ions in discussion but judge it likely that AbPncA functions in the presence of different divalent cations. (Experimental details, including enzyme activity and metal-ion identification, together with sequence alignments, and additional figures are given as Supporting Information Figure S1–S8). Crystals were obtained in the presence of cacodylate buffer and form II shows dimethylarsinoyl-modified Cys159 in the active site, an artifact of crystallization (Supporting Information, Figure S1, S2). The steric hindrance of this modification precludes full occupancy of pyrazinoic acid such that the final refinement was performed with occupancy 0.8 for pyrazinoic acid, 0.2 for the modified cysteine. Crystal form I has two molecules, form II a single molecule in the asymmetric unit, respectively, with a root-mean-square deviation (r.m.s.d.) derived from least-squares fit of Ca atoms of these three molecules of 0.2 . The structures and the interactions formed by ligands within the active sites are essentially identical and we concentrate on form I, a 1.65 resolution structure with full occupancy ligand (Supporting Information, Figure S3). About 60% of residues form elements of secondary structure, these are eight a-helices and nine b-strands (Figure 1, and Supporting Information, Figure S4). The core of the subunit is a parallel b-sheet of strands 1, 2, 5–9. Three helices (a5, a6, a7) lie on one side of the sheet, with a2 placed against the other. A subdomain is placed at

The crystal structure of Leishmania major N5,N10-methylenetetrahydrofolate dehydrogenase/cyclohydrolase and assessment of a potential drug target

Graphical abstract The crystal structure of Leishmania major N5,N10-methylenetetrahydrofolate dehydrogenase/N5,N10-methenyltetrahydrofolate cyclohydrolase is used to assess the potential of this bifunctional enzyme as a drug target. Highlights ► We report the structure of Leishmania major methylenetetrahydrofolate dehydrogenase/cyclohydrolase. ► Sequence–structure comparisons are carried out with homologues from kinetoplastids and the human host. ► The potential of this bifunctional enzyme as a drug target is assessed. ► The similarities between parasite and human enzymes suggest a difficult target for drug discovery.

Structure of Staphylococcus aureus adenylo­succinate lyase (PurB) and assessment of its potential as a target for structure-based inhibitor discovery

The 2.5 Å resolution structure of S. aureus adenylosuccinate lyase is reported and compared with those of orthologues to assess its potential as a template for early stage drug discovery. AMP and a putative assignment of oxalate, the latter an artefact possibly arising from an impurity in the PEG used for crystallization, occupy the active site.

Geobacillus Stearothermophilus 6-Phosphogluconate Dehydrogenase, Complexed with 6-Phosphogluconate.

The structure of 6-phosphogluconate dehydrogenase from a moderate thermophile, G. stearothermophilus, is presented and compared with those of orthologous enzymes.

Crystal Structures of Toxoplasma Gondii Pterin-4A-Carbinolamine Dehydratase and Comparisons with Mammalian and Parasite Orthologues.

The enzyme pterin-4a-carbinolamine dehydratase (PCD) is important for the recycling of pterins within eukaryotic cells. A recombinant expression system for PCD from the apicomplexan parasite Toxoplasma gondii has been prepared, the protein purified and crystallised. Single crystal X-ray diffraction methods have produced a high-resolution structure (1.6A) of the apo-enzyme and a low-resolution structure (3.1A) of a complex with a substrate-like ligand dihydrobiopterin (BH(2)). Analysis of the hydrogen bonding interactions that contribute to binding BH(2) suggest that the ligand is present in an enol tautomeric state, which makes it more similar to the physiological substrate. The enzyme can process (R)- and (S)-forms of pterin-4a-carbinolamine and the ligand complex suggests that His61 and His79 are placed to act independently as general bases for catalysis of the individual enantiomers. Comparisons with orthologues from other protozoan parasites (Plasmodium falciparum and Leishmania major) and with rat PCD, for which the structure is known, indicate a high degree of sequence and structure conservation of this enzyme. The molecular determinants of ligand recognition and PCD reactivity are therefore highly conserved across species.

Initiating a crystallographic analysis of recombinant (S)-2-hydroxypropylphosphonic acid epoxidase from Streptomyces wedmorensis

The oxirane (1R,2S)-1,2-epoxypropylphosphonic acid (fosfomycin) is a natural product antibiotic produced in Streptomyces wedmorensis by the metal-ion-dependent (S)-2-hydroxypropylphosphonic acid epoxidase. This epoxidase is highly unusual since it has no requirement for a haem prosthetic group. The gene encoding the enzyme, fom4, has been cloned and a highly efficient recombinant source of the enzyme established. Two different crystal forms, tetragonal and hexagonal, have been obtained. The hexagonal form displays symmetry consistent with space group P6(1/5)22 and unit-cell parameters a = 86.44, c = 221.56 A, gamma = 120 degrees. The Matthews coefficient, VM, of 2.7 A3 Da(-1) corresponds to two subunits, each of approximate weight 21.4 kDa, in the asymmetric unit with 55% solvent content. These crystals diffract to high resolution and experimental phases are being sought to determine the structure.