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

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

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

Abstract The rotary ATPase family comprises the ATP synthase (F-ATPase), vacuolar ATPase (V-ATPase) and archaeal ATPase (A-ATPase). These either predominantly utilize a proton gradient for ATP synthesis or use ATP to produce a proton gradient, driving secondary transport and acidifying organelles. With advances in 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-ATPases, the secondary structure organization 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

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

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

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

Unexpected structures formed by the kinase RET C634R mutant extracellular domain suggest potential oncogenic mechanisms in MEN2A

The RET receptor tyrosine kinase plays a pivotal role in cell survival, proliferation, and differentiation, and its abnormal activation leads to cancers through receptor fusions or point mutations. Mutations that disrupt the disulfide network in the extracellular domain (ECD) of RET drive multiple endocrine neoplasia type 2A (MEN2A), a hereditary syndrome associated with the development of thyroid cancers. However, structural details of how specific mutations affect RET are unclear. Here, we present the first structural insights into the ECD of the RET(C634R) mutant, the most common mutation in MEN2A. Using electron microscopy, we demonstrate that the C634R mutation causes ligand-independent dimerization of the RET ECD, revealing an unusual tail-to-tail conformation that is distinct from the ligand-induced signaling dimer of WT RET. Additionally, we show that the RETC634R ECD dimer can form complexes with at least two of the canonical RET ligands and that these complexes form very different structures than WT RET ECD upon ligand binding. In conclusion, this structural analysis of cysteine-mutant RET ECD suggests a potential key mechanism of cancer induction in MEN2A, both in the absence and presence of its native ligands, and may offer new targets for therapeutic intervention.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

Sample deposition onto cryo-EM grids: from sprays to jets and back

Despite the great strides made in the field of single-particle cryogenic electron microscopy (cryo-EM) in microscope design, direct electron detectors and new processing suites, the area of sample preparation is still far from ideal. Traditionally, sample preparation involves blotting, which has been used to achieve high resolution, particularly for well behaved samples such as apoferritin. However, this approach is flawed since the blotting process can have adverse effects on some proteins and protein complexes, and the long blot time increases exposure to the damaging air-water interface. To overcome these problems, new blotless approaches have been designed for the direct deposition of the sample on the grid. Here, different methods of producing droplets for sample deposition are compared. Using gas dynamic virtual nozzles, small and high-velocity droplets were deposited on cryo-EM grids, which spread sufficiently for high-resolution cryo-EM imaging. For those wishing to pursue a similar approach, an overview is given of the current use of spray technology for cryo-EM grid preparation and areas for enhancement are pointed out. It is further shown how the broad aspects of sprayer design and operation conditions can be utilized to improve grid quality reproducibly