ADP ribosylation factor-like (ARL) proteins are small GTPases that undergo conformational changes upon nucleotide binding, and which regulate the affinity of ARLs for binding other proteins, lipids or membranes. There is a paucity of structural data on this family of proteins in the Kinetoplastida, despite studies implicating them in key events related to vesicular transport and regulation of microtubule dependent processes. The crystal structure of Leishmania major ARL1 in complex with GDP has been determined to 2.1 Å resolution and reveals a high degree of structural conservation with human ADP ribosylation factor 1 (ARF1). Putative L. major and Trypanosoma brucei ARF/ARL family members have been classified based on structural considerations, amino acid sequence conservation combined with functional data on Kinetoplastid and human orthologues. This classification may guide future studies designed to elucidate the function of specific family members.
ADP ribosylation factor-like; GTPase; Leishmania; protein structure
MenH (2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase) is a key enzyme in the biosynthesis of menaquinone, catalyzing an unusual 2,5-elimination of pyruvate from 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate.
The crystal structure of Staphylococcus aureus MenH has been determined at 2 Å resolution. In the absence of a complex to inform on aspects of specificity a model of the enzyme-substrate complex has been used in conjunction with previously published kinetic analyses, site-directed mutagenesis studies and comparisons with orthologues to investigate the structure and reactivity of MenH.
The overall basic active site displays pronounced hydrophobic character on one side and these properties complement those of the substrate. A complex network of hydrogen bonds involving well-ordered water molecules serves to position key residues participating in the recognition of substrate and subsequent catalysis. We propose a proton shuttle mechanism, reliant on a catalytic triad consisting of Ser89, Asp216 and His243. The reaction is initiated by proton abstraction from the substrate by an activated Ser89. The propensity to form a conjugated system provides the driving force for pyruvate elimination. During the elimination, a methylene group is converted to a methyl and we judge it likely that His243 provides a proton, previously acquired from Ser89 for that reduction. A conformational change of the protonated His243 may be encouraged by the presence of an anionic intermediate in the active site.
The first committed step in the classical biosynthetic route to menaquinone (vitamin K2) is a Stetter-like conjugate addition of α-ketoglutarate with isochorismate. This reaction is catalyzed by the thiamine diphosphate and metal-ion-dependent 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate synthase (MenD). The medium-resolution (2.35 Å) crystal structure of Bacillus subtilis MenD with cofactor and Mn2+ has been determined. Based on structure–sequence comparisons and modeling, a two-stage mechanism that is primarily driven by the chemical properties of the cofactor is proposed. Hypotheses for the molecular determinants of substrate recognition were formulated. Five basic residues (Arg32, Arg106, Arg409, Arg428, and Lys299) are postulated to interact with carboxylate and hydroxyl groups to align substrates for catalysis in combination with a cluster of non-polar residues (Ile489, Phe490, and Leu493) on one side of the active site. The powerful combination of site-directed mutagenesis, where each of the eight residues is replaced by alanine, and steady-state kinetic measurements has been exploited to address these hypotheses. Arg409 plays a significant role in binding both substrates while Arg428 contributes mainly to binding of α-ketoglutarate. Arg32 and in particular Arg106 are critical for recognition of isochorismate. Mutagenesis of Phe490 and Ile489 has the most profound influence on catalytic efficiency, indicating that these two residues are important for binding of isochorismate and for stabilizing the cofactor position. These data allow for a detailed description of the structure–reactivity relationship that governs MenD function and refinement of the model for the catalytic intermediate that supports the Stetter-like conjugate addition.
CoA, coenzyme A; PDB, Protein Data Bank; SAD, single-wavelength anomalous diffraction; SEPHCHC, 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate; SeMet, selenomethionine; ThDP, thiamine diphosphate; PEG, polyethylene glycol; crystal structure; enzyme mechanism; menaquinone biosynthesis; thiamine diphosphate cofactor
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.
The medium-resolution structure of adenylosuccinate lyase (PurB) from the bacterial pathogen Staphylococcus aureus in complex with AMP is presented. Oxalate, which is likely to be an artifact of crystallization, has been modelled in the active site and occupies a position close to that where succinate is observed in orthologous structures. PurB catalyzes reactions that support the provision of purines and the control of AMP/fumarate levels. As such, the enzyme is predicted to be essential for the survival of S. aureus and to be a potential therapeutic target. Comparisons of this pathogen PurB with the enzyme from Escherichia coli are presented to allow discussion concerning the enzyme mechanism. Comparisons with human PurB suggest that the close similarity of the active sites would make it difficult to identify species-specific inhibitors for this enyme. However, there are differences in the way that the subunits are assembled into dimers. The distinct subunit–subunit interfaces may provide a potential area to target by exploiting the observation that creation of the enzyme active site is dependent on oligomerization.
adenylosuccinate lyase; AMP; oxalate; purine biosynthesis; purine cycle
Psychological stress can contribute to health disparities in populations that are confronted with the recurring stress of everyday life. A number of biomarkers have been shown to be affected by psychological stress. These biomarkers include allostatic load, which is a summary measure of the cumulative biological burden of the repeated attempts to adapt to daily stress. Allostatic load includes effects on the hypothalamic-pituitary axis, the sympathetic nervous system and the cardiovascular system. These in turn affect the immune system via bidirectional signaling pathways. Evidence is also building that psychological stress, perhaps via heightened inflammatory states, can increase oxidative stress levels and DNA damage. The inter-relationships of ethnicity, genotype, gene expression and ability to adequately mitigate stress response are just starting to be appreciated. The need to conduct these studies in disadvantaged populations is clear and requires methods to address potential logistical barriers. Biomarkers can help characterize and quantify the biological impact of psychological stress on the etiology of health disparities.
Psychosocial stress; health disparities; allostatic load; oxidative stress
The nonmevalonate route to isoprenoid biosynthesis is essential in Gram-negative bacteria and apicomplexan parasites. The enzymes of this pathway are absent from mammals, contributing to their appeal as chemotherapeutic targets. One enzyme, 2C-methyl-d-erythritol-2,4-cyclodiphosphate synthase (IspF), has been validated as a target by genetic approaches in bacteria. Virtual screening against Escherichia coli IspF (EcIspF) was performed by combining a hierarchical filtering methodology with molecular docking. Docked compounds were inspected and 10 selected for experimental validation. A surface plasmon resonance assay was developed and two weak ligands identified. Crystal structures of EcIspF complexes were determined to support rational ligand development. Cytosine analogues and Zn2+-binding moieties were characterized. One of the putative Zn2+-binding compounds gave the lowest measured KD to date (1.92 ± 0.18 μM). These data provide a framework for the development of IspF inhibitors to generate lead compounds of therapeutic potential against microbial pathogens.
Gas-phase photoelectron spectroscopy and density functional theory have been used to investigate the electronic structures of open-shell bent vanadocene compounds with chelating dithiolate ligands, which are minimum molecular models of the active sites of pyranopterin Mo/W enzymes. The compounds Cp2V(dithiolate) [where dithiolate is 1,2-ethenedithiolate (S2C2H2) or 1,2-benzenedithiolate (bdt), and Cp is cyclopentadienyl] provide access to a 17-electron, d1 electron configuration at the metal center. Comparison with previously studied Cp2M(dithiolate) complexes, where M is Ti and Mo (respectively d0 and d2 electron configurations), allows evaluation of d0, d1 and d2 electronic configurations of the metal-center that are analogues for the metal oxidation states present throughout the catalytic cycle of these enzymes. A “dithiolate-folding effect” that involves an interaction between the vanadium d orbitals and sulfur p orbitals is shown to stabilize the d1 metal center, allowing the d1 electron configuration and geometry to act as a low energy electron pathway intermediate between the d0 and d2 electron configurations of the enzyme.
The social status of groups is key to determining health vulnerability at the population level. The impact of material and psychological stresses imposed by social inequities and marginalization is felt most intensely during perinatal/early childhood and puberty/adolescent periods, when developmental genes are expressed and interact with social-physical environments. The influence of chronic psychosocial stresses on gene expression via neuroendocrine regulatory dysfunction is crucial to understanding the biological bases of adult health vulnerability. Studying childhood biology vulnerabilities to neighborhood environments will aid the crafting of multifaceted, multilevel public policy interventions providing immediate benefits and compounded long-term population health yields.
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
The structure of UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) in complex with UDP is reported. The complex allows for a description of how the enzyme recognizes and binds a nucleotide moiety and enables the construction of an LpxC-substrate model.
The structure of recombinant Aquifex aeolicus UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) in complex with UDP has been determined to a resolution of 2.2 Å. Previous studies have characterized the binding sites of the fatty-acid and sugar moieties of the substrate, UDP-(3-O-hydroxymyristoyl)-N-acetylglucosamine, but not that of the nucleotide. The uracil-binding site is constructed from amino acids that are highly conserved across species. Hydrophobic associations with the Phe155 and Arg250 side chains in combination with hydrogen-bonding interactions with the main chain of Glu154 and the side chains of Tyr151 and Lys227 position the base. The phosphate and ribose groups are directed away from the active site and interact with Arg137, Lys156, Glu186 and Arg250. The orientation of the phosphate-ribose tail is not conducive to catalysis, perhaps owing to the position of an inhibitory Zn2+. However, based on the position of uracil revealed in this study and on the previously reported complex of LpxC with an inhibitor, a model is proposed for substrate binding.
lipid A; Aquifex aeolicus; LpxC
The protozoan Trypanosoma brucei has a functional pteridine reductase (TbPTR1), an NADPH-dependent short-chain reductase that participates in the salvage of pterins, which are essential for parasite growth. PTR1 displays broad-spectrum activity with pterins and folates, provides a metabolic bypass for inhibition of the trypanosomatid dihydrofolate reductase and therefore compromises the use of antifolates for treatment of trypanosomiasis. Catalytic properties of recombinant TbPTR1 and inhibition by the archetypal antifolate methotrexate have been characterized and the crystal structure of the ternary complex with cofactor NADP+ and the inhibitor determined at 2.2 Å resolution. This enzyme shares 50% amino acid sequence identity with Leishmania major PTR1 (LmPTR1) and comparisons show that the architecture of the cofactor binding site, and the catalytic centre are highly conserved, as are most interactions with the inhibitor. However, specific amino acid differences, in particular the placement of Trp221 at the side of the active site, and adjustment of the β6-α6 loop and α6 helix at one side of the substrate-binding cleft significantly reduce the size of the substrate binding site of TbPTR1 and alter the chemical properties compared with LmPTR1. A reactive Cys168, within the active site cleft, in conjunction with the C-terminus carboxyl group and His267 of a partner subunit forms a triad similar to the catalytic component of cysteine proteases. TbPTR1 therefore offers novel structural features to exploit in the search for inhibitors of therapeutic value against African trypanosomiasis.
The twin-arginine translocation (Tat) pathway is a protein
targeting system found in bacteria, archaea, and chloroplasts. Proteins
are directed to the Tat translocase by N-terminal signal peptides
containing SRRxFLK “twin-arginine” amino acid motifs.
The key feature of the Tat system is its ability to transport fully
folded proteins across ionically sealed membranes. For this reason
the Tat pathway has evolved for the assembly of extracytoplasmic redox
enzymes that must bind cofactors, and so fold, prior to export. It
is important that only cofactor-loaded, folded precursors are presented
for export, and cellular processes have been unearthed that regulate
signal peptide activity. One mechanism, termed “Tat proofreading”,
involves specific signal peptide binding proteins or chaperones. The
archetypal Tat proofreading chaperones belong to the TorD family,
which are dedicatedto the assembly of molybdenum-dependent redox
enzymes in bacteria. Here, a gene cluster was identified in the archaeon Archaeoglobus fulgidusthat is predicted to encode a putative
molybdenum-dependent tetrathionate reductase. The gene cluster also
encodes a TorD family chaperone (AF0160 or TtrD) and in this work
TtrD is shown to bind specifically to the Tat signal peptide of the
TtrA subunit of the tetrathionate reductase. In addition, the 3D crystal
structure of TtrD is presented at 1.35 Å resolution and a nine-residue
binding epitope for TtrD is identified within the TtrA signal peptide
close to the twin-arginine targeting motif. This work suggests that
archaea may employ a chaperone-dependent Tat proofreading system that
is similar to that utilized by bacteria.