Alice Dawson

Use of a CCD diffractometer in crystal structure determinations at high pressure

Although CCD instruments are now widely used in single-crystal diffraction, they have not been employed so extensively in crystallographic studies at high pressure. This paper describes some practical experience in the application of one CCD instrument, the Bruker–Nonius SMART APEX (a fixed-χ instrument). Centring a sample in a pressure cell is complicated by the restrictions on viewing the sample imposed by the body of the cell. The data collection strategy is defined by the requirements that (i) the incident beam must illuminate the sample and (ii) no more than 80% of the detector should be shaded by the body of the pressure cell. High-pressure diffraction images are contaminated by powder lines from the gasket and backing-disk materials, which form part of the pressure cell, and very intense spots from the diamond anvils. Procedures for the selection of spots for indexing are described. Integration routines attempt to harvest intensity data from regions of the detector that are shaded by the body of the pressure cell, and a procedure for generating dynamic masks is described. Shading also reduces the volume of reciprocal space that can be sampled, although this can be increased by performing data collections at more than one pressure-cell setting. Corrections for absorption are carried out in a two-stage procedure comprising an analytical correction for absorption by the cell, followed by a second multi-scan correction. Data sets collected at high pressure often contain some significant outliers; these can be identified during merging using a robust resistant weighting scheme, as described by Blessing [J. Appl. Cryst. (1997), 30, 421–426].

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

Induced-fit recognition of DNA by organometallic complexes with dynamic stereogenic centers

Organometallic chemistry offers novel concepts in structural diversity and molecular recognition that can be used in drug design. Here, we consider DNA recognition by η6-arene Ru(II) anticancer complexes by an induced-fit mechanism. The stereochemistry of the dinuclear complex [((η6-biphenyl)RuCl(en))2-(CH2)6]2 + (3, en = ethylenediamine) was elucidated by studies of the half unit [(η6-biphenyl)RuCl(Et-en)]+ (2, where Et-en is Et(H)NCH2CH2NH2). The structures of the separated \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}R_{{\mathrm{Ru}}}^{*}R_{{\mathrm{N}}}^{*}\end{equation*}\end{document} and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}S_{{\mathrm{Ru}}}^{*}R_{{\mathrm{N}}}^{*}\end{equation*}\end{document} diastereomers of 2 were determined by x-ray crystallography; their slow interconversion in water (t½ ≈ 2 h, 298 K, pH 6.2) was observed by NMR spectroscopy. For 2 and 3 the \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}R_{{\mathrm{Ru}}}^{*}R_{{\mathrm{N}}}^{*}\end{equation*}\end{document} configurations are more stable than \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}S_{{\mathrm{Ru}}}^{*}R_{{\mathrm{N}}}^{*}\end{equation*}\end{document} (73:27). X-ray and NMR studies showed that reactions of 2 and 3 with 9-ethylguanine gave rise selectively to \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}S_{{\mathrm{Ru}}}^{*}R_{{\mathrm{N}}}^{*}\end{equation*}\end{document} diastereomers. Dynamic chiral recognition of guanine can lead to high diastereoselectivity of DNA binding. The dinuclear complex 3 induced a large unwinding (31°) of plasmid DNA, twice that of mononuclear 2 (14°), and effectively inhibited DNA-directed RNA synthesis in vitro. This dinuclear complex gave rise to interstrand cross-links on a 213-bp plasmid fragment with efficiency similar to bifunctional cisplatin, and to 1,3-GG interstrand and 1,2-GG and 1,3-GTG intrastrand cross-links on site-specifically ruthenated 20-mers. Complex 3 blocked intercalation of ethidium considerably more than mononuclear 2. The concept of induced-fit recognition of DNA by organometallic complexes containing dynamic stereogenic centers via dynamic epimerization, intercalation, and cross-linking may be useful in the design of anticancer drugs.

Synthesis, structure and properties of [Pt(2,2 '-bipyridyl-5,5 '-dicarboxylic acid)(3,4-toluenedithiolate)]: tuning molecular properties for application in dye-sensitised solar cells

The platinum diimine dithiolate complex, [Pt(2,2′-bipyridyl-5,5′-dicarboxylicacid)(3,4-toluenedithiolate)] ([Pt(5,5′-dcbpy)(tdt)]) and its tetrabutylammonium salt [TBA]2[Pt(5,5′-dcbpy)(tdt)] have been prepared, spectroscopically and electrochemically characterised and attached on to TiO2 substrate to be used as solar cell sensitisers. A single-crystal X-ray structure was obtained for [TBA]2[Pt(5,5′-dcbpy)(tdt)]·EtOH·EtOAc. The effect of the position of the two carboxylic acid substituents on the electrochemistry of the 5,5′-disubstituted complexes is discussed in comparison with the previously reported [Pt(4,4′-dcbpy)(tdt)]. Electrochemical studies show no major change in the HOMO after movement of the carboxylic acid groups, consistent with assignment of the HOMO as largely dithiolate based. Movement of the carboxylic acid groups makes the diimine electronic character and hence the LUMO of the complexes different. Electrochemical studies show a change to lower energy of the LUMO represented by changes in reduction potential of the compound on moving the carboxylic acid substituents from the 4,4′ to the 5,5′ positions. Both [Pt(5,5′-dcbpy)(tdt)] and [TBA]2[Pt(5,5′-dcbpy)(tdt)] have been used as solar cell sensitisers, with the di-TBA salt giving lower dye loading but superior photovoltaic performance. The consequences of tuning the complex through the position of the carboxylic acid groups are discussed.

Structure and reactivity of LpxD, the N-acyltransferase of lipid A biosynthesis

The external layer of the Gram-negative bacterial outer membrane is primarily composed of a protective, selectively permeable LPS. The biosynthesis of LPS relies on UDP-3-O-acyl-glucosamine N-acyltransferase (LpxD), which transfers 3-hydroxy-arachidic acid from acyl carrier protein to the 2′ amine of UDP-3-O-myristoyl glucosamine in Chlamydia trachomatis. Our crystallographic study reveals that LpxD is a homotrimer, each subunit of which is constructed from a novel combination of an N-terminal uridine-binding domain, a core lipid-binding domain, and a C-terminal helical extension. Highly conserved residues dominate nucleotide binding. Phe-43 and Tyr-49 form π-stacking interactions with uracil, and Asn-46 and His-284 form hydrogen bonds with the phosphate groups. These interactions place the glucosamine moiety at the catalytic center formed by two adjacent subunits. Here His-247 and His-284 contribute to a mechanism involving nucleophilic attack by the amine of one substrate on the carbonyl carbon of an acyl carrier protein thioester conjugate. Serendipitously, our study reveals a fatty acid (FA) binding groove near the catalytic center. MS elucidated the presence of a FA mixture binding to LpxD, with palmitic acid the most prevalent. The placement of UDP-N-acetylglucosamine and the FA provides details of N-acyltransferase ligand interactions and allows for a description of structure and reactivity at an early stage of LPS assembly.

Isotopic Polymorphism in Pyridine

The ab initio calculation of the relative stabilities of isomers of gas phase molecules rates as one of the outstanding scientific achievements of the twentieth century. Our understanding of the structure of solid state is, by comparison, much less well advanced. The problem is illustrated by the crystal structure of pyridine. Pyridine (C5H5N or ‘h5’ hereafter) is one of the simplest heteroaromatic compounds but its crystal structure (phase h5-I) is unusually complicated, having four independent molecules in the asymmetric unit (Z’ = 4). [1] Price et al. have surveyed the potential for polymorphism in pyridine using ab initio crystal structure prediction methods, finding over a dozen crystal structures that were energetically competitive with h5-I. [2] In parallel with Price’s theoretical work an intense experimental search was made by one of us (RB group) for new low-temperature polymorphs of pyridine. Though all attempts to crystallize h5 failed to yield anything but the h5-I phase, crystallization of pyridine-d5 (d5) from pentane yielded a new phase, d5-II, at 188 K. Recrystallization from a low-melting solvent such as pentane has been shown in the past to circumvent hightemperature phases because saturation of the solution occurs below the temperature of the phase transition. [3] The new d5-II phase has one molecule in the asymmetric unit (Z’ = 1), but does not correspond to any of the predicted polymorphs of h5. The effect of temperature on the crystal structure of pyridine-d5 was subsequently investigated further using neutron powder diffraction. The sample was ground at 77 K [4] and then rapidly cooled to 2 K. The powder pattern was successfully modelled as d5-I (see Fig. S1a in the Supplementary Information). The sample was then warmed in steps of 2 K, with patterns being acquired at each temperature. When the sample reached 170 K it began to undergo a sluggish phase transition into d5-II. After collecting a clean d5-II neutron powder

Structure and reactivity of Trypanosoma brucei pteridine reductase: inhibition by the archetypal antifolate methotrexate

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.

Pressure-induced polymorphism in phenol

The high-pressure crystal structure of phenol (C(6)H(5)OH), including the positions of the H atoms, has been determined using a combination of single-crystal X-ray diffraction techniques and ab initio density-functional calculations. It is found that at a pressure of 0.16 GPa, which is just sufficient to cause crystallization of a sample held at a temperature just above its ambient-pressure melting point (313 K), a previously unobserved monoclinic structure with P2(1) symmetry is formed. The structure is characterized by the formation of hydrogen-bonded molecular chains, and the molecules within each chain adopt a coplanar arrangement so that they are ordered in an alternating 1-1-1 sequence. Although the crystal structure of the ambient-pressure P112(1) phase is also characterized by the formation of molecular chains, the molecules adopt an approximate threefold arrangement. A series of ab initio calculations indicates that the rearrangement of the molecules from helical to coplanar results in an energy difference of only 0.162 eV molecule(-1) (15.6 kJ mole(-1)) at 0.16 GPa. The calculations also indicate that there is a slight increase in the dipole moment of the molecules, but, as the high-pressure phase has longer hydrogen-bond distances, it is found that, on average, the hydrogen bonds in the ambient-pressure phase are stronger.

Specificity and Reactivity in Menaquinone Biosynthesis: The Structure of Escherichia coli MenD (2-Succinyl-5-Enolpyruvyl-6-Hydroxy-3-Cyclohexadiene-1-Carboxylate Synthase)

The thiamine diphosphate (ThDP) and metal-ion-dependent enzyme 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate synthase, or MenD, catalyze the Stetter-like conjugate addition of alpha-ketoglutarate with isochorismate to release 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate and carbon dioxide. This reaction represents the first committed step for biosynthesis of menaquinone, or vitamin K2, a key cofactor for electron transport in bacteria and a metabolite for posttranslational modification of proteins in mammals. The medium-resolution structure of MenD from Escherichia coli (EcMenD) in complex with its cofactor and Mn2+ has been determined in two related hexagonal crystal forms. The subunit displays the typical three-domain structure observed for ThDP-dependent enzymes in which two of the domains bind and force the cofactor into a configuration that supports formation of a reactive ylide. The structures reveal a stable dimer-of-dimers association in agreement with gel filtration and analytical ultracentrifugation studies and confirm the classification of MenD in the pyruvate oxidase family of ThDP-dependent enzymes. The active site, created by contributions from a pair of subunits, is highly basic with a pronounced hydrophobic patch. These features, formed by highly conserved amino acids, match well to the chemical properties of the substrates. A model of the covalent intermediate formed after reaction with the first substrate alpha-ketoglutarate and with the second substrate isochorismate positioned to accept nucleophilic attack has been prepared. This, in addition to structural and sequence comparisons with putative MenD orthologues, provides insight into the specificity and reactivity of MenD and allows a two-stage reaction mechanism to be proposed.

Crystal structures of IspF from Plasmodium falciparum and Burkholderia cenocepacia: comparisons inform antimicrobial drug target assessment

Background2C-methyl-D-erythritol-2,4-cyclodiphosphate synthase (IspF) catalyzes the conversion of 4-diphosphocytidyl-2C-methyl-D-erythritol-2-phosphate to 2C-methyl-D-erythritol-2,4-cyclodiphosphate and cytidine monophosphate in production of isoprenoid-precursors via the methylerythritol phosphate biosynthetic pathway. IspF is found in the protozoan Plasmodium falciparum, a parasite that causes cerebral malaria, as well as in many Gram-negative bacteria such as Burkholderia cenocepacia. IspF represents a potential target for development of broad-spectrum antimicrobial drugs since it is proven or inferred as essential in these pathogens and absent from mammals. Structural studies of IspF from these two important yet distinct pathogens, and comparisons with orthologues have been carried out to generate reagents, to support and inform a structure-based approach to early stage drug discovery.ResultsEfficient recombinant protein production and crystallization protocols were developed, and high-resolution crystal structures of IspF from P. falciparum (Pf IspF) and B. cenocepacia (Bc IspF) in complex with cytidine nucleotides determined. Comparisons with orthologues, indicate a high degree of order and conservation in parts of the active site where Zn2+ is bound and where recognition of the cytidine moiety of substrate occurs. However, conformational flexibility is noted in that area of the active site responsible for binding the methylerythritol component of substrate. Unexpectedly, one structure of Bc IspF revealed two molecules of cytidine monophosphate in the active site, and another identified citrate coordinating to the catalytic Zn2+. In both cases interactions with ligands appear to help order a flexible loop at one side of the active site. Difficulties were encountered when attempting to derive complex structures with other ligands.ConclusionsHigh-resolution crystal structures of IspF from two important human pathogens have been obtained and compared to orthologues. The studies reveal new data on ligand binding, with citrate coordinating to the active site Zn2+ and when present in high concentrations cytidine monophosphate displays two binding modes in the active site. Ligand binding appears to order a part of the active site involved in substrate recognition. The high degree of structural conservation in and around the IspF active site suggests that any structural model might be suitable to support a program of structure-based drug discovery.