The effect of dolichol on the structure and phase behaviour of phospholipid model membranes

The effect of dolichol C₉₅ on the structure and thermotropic phase behaviour of dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine and stearoyloleoylphosphatidylethanolamine has been examined by synchrotron X-ray diffraction and differential scanning calorimetry. The presence of dolichol C₉₅ had no detectible effects on the temperature of either the gel to ripple or the ripple to liquid-crystal phase transition of dipalmitoylphosphatidylcholine. A proportionate increase of a few degrees in the temperature of the gel to lamellar liquid-crystal phase transition is observed in dispersions of dipalmitoylphosphatidylethanolamine and significantly there is a decrease in the temperature of the lamellar to non-lamellar phase transition of stearoyloleoylphosphatidylethanolamine. There was no significant change in the bilayer repeat spacing of all three mixed dispersions in gel phase in the presence of up to 20 mol% dolichol C₉₅. Electron density calculations showed that there was no change of bilayer thickness of dipalmitoylphosphatidylcholine with incorporation of up to 7.5 mol% dolichol C₉₅. These data suggest that effect of dolichol on the phospholipid model membranes depend on both the head group and the hydrocarbon chains of the phospholipid molecules. The presence of dolichol in phosphatidylcholine bilayers conforms to a model in which the polyisoprene compound is phase separated into a central domain sandwiched between the two monolayers in gel phase. In bilayers of phosphatidylethanolamines dolichol tends to stabilize the bilayers in gel phase at low temperatures and destabilize the bilayers in lamellar disordered structure at high temperatures. Non-lamellar structures coexist with lamellar disordered phase over a wide temperature range suggesting that dolichol is enriched in domains of non-lamellar structure and depleted from lamellar phase. These findings are useful to understand the function of dolichol in cell membranes.     

Crystal structure of the parasite protease inhibitor chagasin in complex with a host target cysteine protease

Chagasin is a protein produced by Trypanosoma cruzi, the parasite that causes Chagas' disease. This small protein belongs to a recently defined family of cysteine protease inhibitors. Although resembling well-known inhibitors like the cystatins in size (110 amino acid residues) and function (they all inhibit papain-like (C1 family) proteases), it has a unique amino acid sequence and structure. We have crystallized and solved the structure of chagasin in complex with the host cysteine protease, cathepsin L, at 1.75 A resolution. An inhibitory wedge composed of three loops (L2, L4, and L6) forms a number of contacts responsible for high-affinity binding (K(i), 39 pM) to the enzyme. All three loops interact with the catalytic groove, with the central loop L2 inserted directly into the catalytic center. Loops L4 and L6 embrace the enzyme molecule from both sides and exhibit distinctly different patterns of protein-protein recognition. Comparison with a 1.7 A structure of uncomplexed chagasin, also determined in this study, demonstrates that a conformational change of the first binding loop (L4) allows extended binding to the non-primed substrate pockets of the enzyme active site cleft, thereby providing a substantial part of the inhibitory surface. The mode of chagasin binding is generally similar, albeit distinctly different in detail, when compared to those displayed by cystatins and the cysteine protease inhibitory p41 fragment of the invariant chain. The chagasin-cathepsin L complex structure provides details of how the parasite protein inhibits a host enzyme of possible importance in host defense. The high level of structural and functional similarity between cathepsin L and the T. cruzi enzyme cruzipain gives clues to how the cysteine protease activity of the parasite can be targeted. This information will aid in the development of synthetic inhibitors for use as potential drugs for the treatment of Chagas disease.     

Structure of an amide bond forming F(420):gamma-glutamyl ligase from Archaeoglobus fulgidus -- a member of a new family of non-ribosomal peptide synthases

F(420) is a flavin-like redox-active coenzyme commonly used by archaea and some eubacteria in a variety of biochemical reactions in methanogenesis, the formation of secondary metabolites, the degradation of nitroaromatic compounds, activation of nitroimidazofurans, and F(420)-dependent photolysis in DNA repair. Coenzyme F(420)-2 biosynthesis from 7,8-didemethyl-8-hydroxy-5-deazariboflavin (Fo) and lactaldehyde involves six enzymatic steps and five proteins (CofA, CofB, CofC, CofD, and CofE). CofE, a F(420)-0:gamma-glutamyl ligase, is responsible for the last two enzymatic steps; it catalyses the GTP-dependent addition of two L-glutamate residues to F(420)-0 to form F(420)-2. CofE is found in archaea, the aerobic actinomycetes, and cyanobacteria. Here, we report the first crystal structure of the apo-F(420)-0:gamma-glutamyl ligase (CofE-AF) from Archaeoglobus fulgidus and its complex with GDP at 2.5 A and 1.35 A resolution, respectively. The structure of CofE-AF reveals a novel protein fold with an intertwined, butterfly-like dimer formed by two-domain monomers. GDP and Mn(2+) are bound within the putative active site in a large groove at the dimer interface. We show that the enzyme adds a glutamate residue to both F(420)-0 and F(420)-1 in two distinct steps. CofE represents the first member of a new structural family of non-ribosomal peptide synthases.     

Structure and action of a C-C bond cleaving alpha/beta-hydrolase involved in nicotine degradation

The enzyme 2,6-dihydroxy-pseudo-oxynicotine hydrolase from the nicotine-degradation pathway of Arthrobacter nicotinovorans was crystallized and the structure was determined by an X-ray diffraction analysis at 2.1 A resolution. The enzyme belongs to the alpha/beta-hydrolase family as derived from the chain-fold and from the presence of a catalytic triad with its oxyanion hole at the common position. This relationship assigns a pocket lined by the catalytic triad as the active center. The asymmetric unit contains two C(2)-symmetric dimer molecules, each adopting a specific conformation. One dimer forms a more spacious active center pocket and the other a smaller one, suggesting an induced-fit. All of the currently established C-C bond cleaving alpha/beta-hydrolases are from bacterial meta-cleavage pathways for the degradation of aromatic compounds and cover their active center with a 40 residue lid placed between two adjacent strands of the beta-sheet. In contrast, the reported enzyme shields its active center with a 110 residue N-terminal domain, which is absent in the meta-cleavage hydrolases. Since neither the substrate nor an analogue could be bound in the crystals, the substrate was modeled into the active center using the oxyanion hole as a geometric constraint. The model was supported by enzymatic activity data of 11 point mutants and by the two dimer conformations suggesting an induced-fit. Moreover, the model assigned a major role for the large N-terminal domain that is specific to the reported enzyme. The proposal is consistent with the known data for the meta-cleavage hydrolases although it differs in that the reaction does not release alkenes but a hetero-aromatic compound in a retro-Friedel-Crafts acylation. Because the hydrolytic water molecule can be assigned to a geometrically suitable site that can be occupied in the presence of the substrate, the catalytic triad may not form a covalent acyl-enzyme intermediate but merely support a direct hydrolysis.     

Structural and kinetic characterization of quinolinate phosphoribosyltransferase (hQPRTase) from homo sapiens

Human quinolinate phosphoribosyltransferase (EC 2.4.2.19) (hQPRTase) is a member of the type II phosphoribosyltransferase family involved in the catabolism of quinolinic acid (QA). It catalyses the formation of nicotinic acid mononucleotide from quinolinic acid, which involves a phosphoribosyl transfer reaction followed by decarboxylation. hQPRTase has been implicated in a number of neurological conditions and in order to study it further, we have carried out structural and kinetic studies on recombinant hQPRTase. The structure of the fully active enzyme overexpressed in Escherichia coli was solved using multiwavelength methods to a resolution of 2.0 A. hQPRTase has a alpha/beta barrel fold sharing a similar overall structure with the bacterial QPRTases. The active site of hQPRTase is located at an alpha/beta open sandwich structure that serves as a cup for the alpha/beta barrel of the adjacent subunit with a QA binding site consisting of three arginine residues (R102, R138 and R161) and two lysine residues (K139 and K171). Mutation of these residues affected substrate binding or abolished the enzymatic activity. The kinetics of the human enzyme are different to the bacterial enzymes studied, hQPRTase is inhibited competitively and non-competitively by one of its substrates, 5-phosphoribosylpyrophosphate (PRPP). The human enzyme adopts a hexameric arrangement, which places the active sites in close proximity to each other.     

Quaternary protoberberine alkaloids

This contribution reviews some general aspects of the quaternary iminium protoberberine alkaloids. The alkaloids represent a very extensive group of secondary metabolites with diverse structures, distribution in nature, and biological effects. The quaternary protoberberine alkaloids (QPA), derived from the 5,6-dihydrodibenzo[a,g]quinolizinium system, belong to a large class of isoquinoline alkaloids. Following a general introduction, the plant sources of QPA, their biosynthesis, and procedures for their isolation are discussed. Analytical methods and spectral data are summarized with emphasis on NMR spectroscopy. The reactivity of QPA is characterized by the sensitivity of the iminium bond CN(+) to nucleophilic attack. The addition of various nucleophiles to the protoberberine skeleton is discussed. An extended discussion of the principal chemical reactivity is included since this governs interactions with biological targets. Quaternary protoberberine alkaloids and some related compounds exhibit considerable biological activities. Recently reported structural studies indicate that the QPA interact with nucleic acids predominantly as intercalators or minor groove binders. Currently, investigations in many laboratories worldwide are focused on the antibacterial and antimalarial activity, cytotoxicity, and potential genotoxicity of QPA.     

X-ray diffraction and high-resolution NMR spectroscopy of methyl 3,4-di-O-acetyl-1,5-anhydro-2-deoxy-D-arabino-hex-1-enopyranuronate

Single-crystal X-ray diffraction and high-resolution (1)H and (13)C NMR spectral data for methyl 3,4-di-O-acetyl-1,5-anhydro-2-deoxy-D-arabino-hex-1-enopyranuronate are reported. The (5)H(4) conformation was found to be the preferred form for this glycal, both in the crystal lattice and in solution. The factors determining the (4)H(5)<==>(5)H(4) conformational equilibrium for acetylated glycals are discussed.     

Structure of the Mn<Subscript>4</Subscript>–Ca cluster as derived from X-ray diffraction

The catalytic centre for light-induced water oxidation in photosystem II (PSII) is a multinuclear metal cluster containing four manganese and one calcium cations. Knowing the structure of this biological catalyst is of utmost importance for unravelling the mechanism of water oxidation in photosynthesis. In this review we describe the current state of the X-ray structure determination at 3.0 A resolution of the water oxidation complex (WOC) of PSII. The arrangement of metal cations in the cluster, their coordination and protein surroundings are discussed with regard to spectroscopic and mutagenesis studies. Limitations of the presently available structural data are pointed out and possible perspectives for the future are outlined, including the combination of X-ray diffraction and X-ray spectroscopy on single crystals.     

The structural diversity of DNA–neutral phospholipids–divalent metal cations aggregates: a small-angle synchrotron X-ray diffraction study

We investigate the structure of aggregates formed due to DNA interaction with saturated neutral phosphatidylcholines [dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine] in presence of Ca²⁺ and Mg²⁺ cations using simultaneous synchrotron small- and wide-angle X-ray diffractions. For DPPC:DNA = 3:1 mol/base and in the range of 1–50 mM Ca²⁺, the diffractograms show structural heterogeneity of aggregates. We observe the coexistence of two lamellar phases in aggregates prepared at 1 mM Ca²⁺: L^x phase with the DNA strands (of unknown organization) intercalated in water layers between adjacent lipid bilayers and L_DPPC phase of DPPC bilayers without any divalent cations and DNA strands. Aggregates prepared in the range 2–50 mM Ca²⁺ show a condensed gel lamellar phase L_g^c with the lipid bilayer periodicity d ≈ 8.0 nm, and the DNA–DNA interhelical distance d _DNA ≈ 5.1 nm. The increase of temperature induces the decrease in the intensity and the increase in the width of the DNA related peak. In the fluid state, the condensed lamellar phase L_α^c gradually converts into L^x phase. The aggregates do not exhibit rippled P_β phase. The thermal behaviour of aggregates was investigated in the range 20–80°C. Applying heating–cooling cycles, the aggregates converted into energetically more favourable structure: a condensed lamellar phase L^c (or L^x) is preserved or we observe lateral segregation of the DNA strands and metal cations (L^x phase) in coexistence with L_PC phase of pure phospholipids. Dedicated to Prof. Dr Klaus Arnold on the occasion of his 65th birthday.