Molecular basis for resistance of acanthamoeba tubulins to all major classes of antitubulin compounds

Tubulin is essential to eukaryotic cells and is targeted by several antineoplastics, herbicides, and antimicrobials. We demonstrate that Acanthamoeba spp. are resistant to five antimicrotubule compounds, unlike any other eukaryote studied so far. Resistance correlates with critical amino acid differences within the inhibitor binding sites of the tubulin heterodimers

Using a SMALP platform to determine a sub-nm single particle cryo-EM membrane protein structure

The field of membrane protein structural biology has been revolutionized over the last few years with a number of high profile structures being solved using cryo-EM including Piezo, Ryanodine receptor, TRPV1 and the Glutamate receptor. Further developments in the EM field hold the promise of even greater progress in terms of greater resolution, which for membrane proteins is still typically within the 4-7 angstrom range. One advantage of a cryo-EM approach is the ability to study membrane proteins in more "native" like environments for example proteoliposomes, amphipols and nanodiscs. Recently, styrene maleic acid co-polymers (SMA) have been used to extract membrane proteins surrounded by native lipids (SMALPs) maintaining a more natural environment. We report here the structure of the Escherichia coli multidrug efflux transporter AcrB in a SMALP scaffold to sub-nm resolution, with the resulting map being consistent with high resolution crystal structures and other EM derived maps. However, both the C-terminal helix (TM12) and TM7 are poorly defined in the map. These helices are at the exterior of the helical bundle and form the greater interaction with the native lipids and SMA polymer and may represent a more dynamic region of the protein. This work shows the promise of using an SMA approach for single particle cryo-EM studies to provide sub-nm structures.Peer reviewe

The changing landscape of membrane protein structural biology through developments in electron microscopy

Membrane proteins are ubiquitous in biology and are key targets for therapeutic development. Despite this, our structural understanding has lagged behind that of their soluble counterparts. This review provides an overview of this important field, focusing in particular on the recent resurgence of electron microscopy (EM) and the increasing role it has to play in the structural studies of membrane proteins, and illustrating this through several case studies. In addition we examine some of the challenges remaining in structural determination, and what steps are underway to enhance our knowledge of these enigmatic proteins

The acanthamoeba shikimate pathway has a unique molecular arrangement and is essential for aromatic amino acid biosynthesis

The shikimate pathway is the only known biosynthetic route for de novo synthesis of aromatic compounds. It is described as an ancient eukaryotic innovation that has been retained in a subset of eukaryotes, replaced in plants through the acquisition of the chloroplast, but lost in many including humans. Herein, we demonstrate that Acanthamoeba castellanii possesses the shikimate pathway by biochemical and a combination of bioinformatics and molecular biological methods. The growth of A. castellanii (Neff strain and a recently isolated clinical specimen, both T4 genotypes) is inhibited by glyphosate [N-(phosphonomethyl) glycine], an inhibitor of EPSP synthase and the addition of phenylalanine and tryptophan, which are dependent on the shikimate pathway, rescued A. castellanii from glyphosate indicating that glyphosate was specific in action. A. castellanii has a novel complement of shikimate pathway enzymes including unique gene fusions, two Type I and one Type II DAHP synthases (for which their likely sensitivities to feedback inhibition by phenylalanine, tyrosine and tryptophan has been modelled) and a canonical chorismate synthase. The shikimate pathway in A. castellanii therefore has a novel molecular arrangement, is required for amino acid biosynthesis and represents an attractive target for antimicrobials

Rotating with the brakes on and other unresolved features of the vacuolar ATPase

The rotary ATPase family is comprised of the ATP synthase (F-ATPase), vacuolar ATPase (V-ATPase) and acrahael ATPase (A-ATPase). These either predominantly utilise a proton gradient for ATP synthesis or use ATP to produce a proton gradient, driving secondary transport and acidifying organelles. With advances in electron microscopy (EM) has come a significant increase in our understanding of the rotary ATPase family. Following the sub nm resolution reconstructions of both the F and V-ATPase the secondary structure organisation of the elusive subunit a has now been resolved, revealing a novel helical arrangement. Despite these significant developments in our understanding of the rotary ATPases there are still a number of unresolved questions about the mechanism, regulation, and overall architecture, which this mini-review aims to highlight and discuss

Structural studies of enoyl acyl carrier protein reductase from Plasmodium falciparum and Toxoplasma gondii.

Enoyl acyl carrier protein reductase enzyme (ENR) catalyses one of the two reductive steps in fatty acid elongation within the fatty acid synthesis type II cycle that is common to plants and prokaryotes. Since enzymes of this pathway are absent in humans they have become the target for several potent antibacterial compounds including triclosan which inhibits ENR in the picomolar range. As part of this thesis the gene for a type II ENR was located in the genomes of the apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii. Analysis of the derived protein sequences suggested that these enzyme reside in the apicoplast. X-ray crystallographic techniques have been used to solve the structure for Plasmodium falciparum (Pf) and Toxoplasma gondii (Tg) ENR in complex with the NAD+ cofactor and triclosan by molecular replacement to 2.2A and 2.6A, respectively. Both enzymes. are tetrameric with the approximate dimensions of 90A x 90A x 50A. Each subunit is formed by a 7 stranded parallel β-sheet flanked by 9α helices, reminiscent of a Rossmann nucleotide binding fold common to several NAD+ binding enzymes. Analysis of the ENR family reveals that a characteristic of apicomplexan ENRs is an insert which varies in size from 42 residues in the P jalciparum enzyme to 6 residues in T.gondii ENR and which flanks the inhibitor/substrate binding site. In PfENR this loop is disordered but in the structure of TgENR the loop can be clearly seen and the structure shows that the loop lies close to the bound inhibitor but makes no direct contacts. Comparisons of the binding sites of a range of different ENR inhibitor complexes has led to a better understanding of the plasticity of the enzyme in response to inhibitor (and possibly substrate) binding. Moreover analysis of the substrate/inhibitor binding pocket in P jalciparum and T.gondii ENR shows that whilst they are similar to the bacterial enzymes there are distinct differences which could be exploited for the development of novel antiparasitic agents. A major hurdle in the delivery of inhibitors targeted towards the apicoplast organelle is the need to cross several barriers including the parasite membranes and host cell walls. However the addition of a releasable eight arginine linker to the phenolic OH group of triclosan significantly improved the speed of delivery and enabled triclosan to enter both the extracellular and intracellular T.gondii tachyzoites and bradyzoites. The identification of both a novel inhibitor for the apicomplexan family and a possible general delivery mechanism may provide a foundation for the development of ENR inhibitors that will efficiently treat several key parasitic diseases

NIMBUS: The Near-Infrared Multi-Band Ultraprecise Spectroimager for SOFIA

We present a new and innovative near-infrared multi-band ultraprecise spectroimager (NIMBUS) for SOFIA. This design is capable of characterizing a large sample of extrasolar planet atmospheres by measuring elemental and molecular abundances during primary transit and occultation. This wide-field spectroimager would also provide new insights into Trans-Neptunian Objects (TNO), Solar System occultations, brown dwarf atmospheres, carbon chemistry in globular clusters, chemical gradients in nearby galaxies, and galaxy photometric redshifts. NIMBUS would be the premier ultraprecise spectroimager by taking advantage of the SOFIA observatory and state of the art infrared technologies. This optical design splits the beam into eight separate spectral bandpasses, centered around key molecular bands from 1 to 4 microns. Each spectral channel has a wide field of view for simultaneous observations of a reference star that can decorrelate time-variable atmospheric and optical assembly effects, allowing the instrument to achieve ultraprecise calibration for imaging and photometry for a wide variety of astrophysical sources. NIMBUS produces the same data products as a low-resolution integral field spectrograph over a large spectral bandpass, but this design obviates many of the problems that preclude high-precision measurements with traditional slit and integral field spectrographs. This instrument concept is currently not funded for development.Comment: 14 pages, 9 figures, SPIE Astronomical Telescopes and Instrumentation 201

Elucidating the structural basis for differing enzyme inhibitor potency by cryo-EM

Histidine biosynthesis is an essential process in plants and microorganisms, making it an attractive target for the development of herbicides and antibacterial agents. Imidazoleglycerol-phosphate dehydratase (IGPD), a key enzyme within this pathway, has been biochemically characterized in both Saccharomyces cerevisiae (Sc_IGPD) and Arabidopsis thaliana (At_IGPD). The plant enzyme, having been the focus of in-depth structural analysis as part of an inhibitor development program, has revealed details about the reaction mechanism of IGPD, whereas the yeast enzyme has proven intractable to crystallography studies. The structure–activity relationship of potent triazole-phosphonate inhibitors of IGPD has been determined in both homologs, revealing that the lead inhibitor (C348) is an order of magnitude more potent against Sc_IGPD than At_IGPD; however, the molecular basis of this difference has not been established. Here we have used single-particle electron microscopy (EM) to study structural differences between the At and Sc_IGPD homologs, which could influence the difference in inhibitor potency. The resulting EM maps at ∼3 Å are sufficient to de novo build the protein structure and identify the inhibitor binding site, which has been validated against the crystal structure of the At_IGPD/C348 complex. The structure of Sc_IGPD reveals that a 24-amino acid insertion forms an extended loop region on the enzyme surface that lies adjacent to the active site, forming interactions with the substrate/inhibitor binding loop that may influence inhibitor potency. Overall, this study provides insights into the IGPD family and demonstrates the power of using an EM approach to study inhibitor binding

Conformational changes during human P2X7 receptor activation examined by structural modelling and cysteine-based cross-linking studies

The P2X7 receptor (P2X7R) is important in mediating a range of physiological functions and pathologies associated with tissue damage and inflammation and represents an attractive therapeutic target. However, in terms of their structure-function relationships, the mammalian P2X7Rs remain poorly characterised compared to some of their other P2XR counterparts. In this study, combining cysteine-based cross-linking and whole-cell patch-clamp recording, we examined six pairs of residues (A44/I331, D48/I331, I58/F311, S60/L320, I75/P177 and K81/V304) located in different parts of the extracellular and transmembrane domains of the human P2X7R. These residues are predicted to undergo substantial movement during the transition of the receptor ion channel from the closed to the open state, predictions which are made based on structural homology models generated from the crystal structures of the zebrafish P2X4R. Our results provide evidence that among the six pairs of cysteine mutants, D48C/I133C and K81C/V304C formed disulphide bonds that impaired the channel gating to support the notion that such conformational changes, particularly those in the outer ends of the transmembrane domains, are critical for human P2X7R activation

The Prevention of Delirium and Complications Associated with Surgical Treatments (PODCAST) study: protocol for an international multicentre randomised controlled trial

Introduction: Postoperative delirium is one of the most common complications of major surgery, affecting 10–70% of surgical patients 60 years and older. Delirium is an acute change in cognition that manifests as poor attention and illogical thinking and is associated with longer intensive care unit (ICU) and hospital stay, long-lasting cognitive deterioration and increased mortality. Ketamine has been used as an anaesthetic drug for over 50 years and has an established safety record. Recent research suggests that, in addition to preventing acute postoperative pain, a subanaesthetic dose of intraoperative ketamine could decrease the incidence of postoperative delirium as well as other neurological and psychiatric outcomes. However, these proposed benefits of ketamine have not been tested in a large clinical trial. Methods: The Prevention of Delirium and Complications Associated with Surgical Treatments (PODCAST) study is an international, multicentre, randomised controlled trial. 600 cardiac and major non-cardiac surgery patients will be randomised to receive ketamine (0.5 or 1 mg/kg) or placebo following anaesthetic induction and prior to surgical incision. For the primary outcome, blinded observers will assess delirium on the day of surgery (postoperative day 0) and twice daily from postoperative days 1–3 using the Confusion Assessment Method or the Confusion Assessment Method for the ICU. For the secondary outcomes, blinded observers will estimate pain using the Behavioral Pain Scale or the Behavioral Pain Scale for Non-Intubated Patients and patient self-report. Ethics and dissemination The PODCAST trial has been approved by the ethics boards of five participating institutions; approval is ongoing at other sites. Recruitment began in February 2014 and will continue until the end of 2016. Dissemination plans include presentations at scientific conferences, scientific publications, stakeholder engagement and popular media. Registration details The study is registered at clinicaltrials.gov, NCT01690988 (last updated March 2014). The PODCAST trial is being conducted under the auspices of the Neurological Outcomes Network for Surgery (NEURONS). Trial registration number NCT01690988 (last updated December 2013)