Molecular modelling techniques have been applied to calculate the three-dimensional architecture and the conformational flexibility of a complete bacterial S-form lipopolysaccharide (LPS) consisting of a hexaacyl lipid A identical to Escherichia coli lipid A, a complete Salmonella typhimurium core oligosaccharide portion, and four repeating units of the Salmonella serogroup B O-specific chain. X-ray powder diffraction experiments on dried samples of LPS were carried out to obtain information on the dimensions of the various LPS partial structures. Up to the Ra-LPS structure, the calculated model dimensions were in good agreement with experimental data and were 2.4 nm for lipid A, 2.8 nm for Re-LPS, 3.5 nm for Rd-LPS, and 4.4 nm for Ra-LPS. The maximum length of a stretched S-form LPS model bearing four repeating units was evaluated to be 9.6 nm; however, energetically favored LPS conformations showed the O-specific chain bent with respect to the Ra-LPS portion and significantly smaller dimensions (about 5.0 to 5.5 nm). According to the calculations, the Ra-LPS moiety has an approximately cylindrical shape and is conformationally well defined, in contrast to the O-specific chain, which was found to be the most flexible portion within the molecule.

No abstract

No abstract

Molecular modelling techniques have been applied to compute the conformations accessible to bacterial deep rough lipopolysaccharide of Escherichia coli (Re-LPS). Analyses of the results showed that the models typically exhibit a tilt of the diglucosamine backbone with respect to the membrane normal of 53 7" while both the glucosamine ring planes are orientated approximately parallel to the membrane normal. Different models were found to show compact and elongated types of acyl chain arrangements, both producing anisotropic lateral dimensions of the models of 1.0-1.1 nm and 1.7-2.0 nm for the shorter and the longer side, respectively. The conformationally allowed range of the isolated dOclA(a-2-4)dOclA disaccharide (dOclA = 3-deoxy-~-mannooctulosonic acid) was found to be extremely limited. It appeared that the dOclA disaccharide (dOclA)2 is centred at the top of the Re-LPS molecule preferring two orientations stabilized by hydrogen bonds involving only one phosphate group of the lipid A moiety at a time.The effect of charges on the Re-LPS conformations has been studied in separate calculations. From these calculations it was obvious that charges have no significant effects on the conformations of the isolated lipid A and (dOclA)2 moieties. However, it was found that the orientation of (dOclA), with respect to the lipid A part is highly sensitive to charges, i. e. in the charged models the proximity of phosphate and carboxyl groups is prevented by strong electrostatic repulsion between these negatively charged groups.In order to rationalize the acyl chain packing of the models, a simple geometrical model which correlates the tilt of the diglucosamine backbone with the energetically favoured close packing of the acyl chains is proposed. Furthermore, the possibility of a chelate-like complexation of divalent cations and its contribution to head group mobility is discussed.Lipopolysaccharides (LPS) are the main constituent of the outer leaflet of the outer membrane of Gram-negative bacteria. As LPS are of special significance to bacterial viability and to interactions of bacteria with host organisms, many studies have been focused on the elucidation of their structure and function [l -51. Chemically, LPS consists of a lipid A and a polysaccharide component, of which the latter is usually subdivided into two regions [6], i.e. the 0-specific chain and the core oligosaccharide. Re-LPS is the smallest LPS found in viable Escherichia coli bacteria [2, 71. It consists of the lipid A part and a partial core built up by a dimer of the rare saccharide 3-deoxy-~-manno-octu~osonic acid (dOclA). Recent work has shown that LPS endotoxic activity is determined by structural components as they are present in E. coli lipid A, i.e. a 8-1-6 interlinked diglucosamine, two phosphoryl groups and at least five, but not more than six, fatty acids including one or two 3-acyloxyacyl groups [S]. However, as itCorrespondence to

The dissociation of the highly aggregated form of lipopolysaccharide (LPS) from Gram-negative bacteria to the monomeric (or soluble) form is though to be the initial step in the activation of responding cells (macrophages, B-cells, neutrophils, monocytes, and endothelial cells) by LPS. This process is presently not adequately understood. Using the equilibrium dialysis apparatus and a highly purified and well-characterized radiolabeled deep rough chemotype LPS ([14C]ReLPS) from Escherichia coli D31m4, we have examined the effect of pH on its solubility (CT) and ionic states in aqueous media. The solubility range of [14C]ReLPS suspended in 50 mM Tris-HCl-100 mM KCl buffer (or 50 mM MES-100 mM KCl buffer at pH 6.5) was determined to be from (2.91 +/- 0.01) x 10(-8) to (4.55 +/- 0.07) x 10(-8) M over a pH range of 6.50-8.20, respectively. These experimental data satisfactorily fitted the curve generated by the solubility equation CT = S0(1 + K5/[H+])/([H+]/K4' + 1), where S0 is the concentration of the tetraanionic ReLPS, K5 is the dissociation constant of the tetraanionic ReLPS in solution, and K4' is the dissociation constant of the trianionic ReLPS at the surface of the solid particles in suspension. The increase in solubility of ReLPS with increase in pH from 7.00 to 8.20 is primarily caused by the formation of the pentaanionic form from the tetraanions. The pK5 (primarily the second dissociation of the 1-phosphate) of ReLPS was determined to be 8.58 from experimental data.(ABSTRACT TRUNCATED AT 250 WORDS)

Six previously published conformational models of Escherichia coli Re lipopolysaccharide (ReLPS) were subjected to molecular dynamics simulations using the CHARMM force field. The monomers of ReLPS were completely immersed in a water box. The dynamic behavior of the solvated models in the presence and absence of calcium cations was compared. The structure of the solvent shell was analyzed in terms of radial distribution functions. Diffusion coefficients and mean residence times were analyzed to characterize the dynamic behavior of the solvent. Order parameters and number of gauche defects were used for the description of the dynamics of the acyl chains. The cations are preferentially located between the carboxylate and phosphate groups of the headgroup. Their presence leads to a rigidification of the headgroup structure and alters the conformation of the backbone, thus influencing the structure and flexibility of the hydrophobic region as well. The effect of calcium on the backbone flexibility was measured in terms of glycosidic torsion angles. The six fatty acid chains of each ReLPS monomer adopt a highly ordered micromembrane structure. The packing parameter indicates that aggregation of these ReLPS monomers will lead to lamellar structures. Evaluation of all data enables us to present one conformation, C, which is thought to best represent the average structure of the ReLPS conformers.

X-ray powder-diffraction experiments have been performed on dry samples of lipid A and various rough-mutant lipopolysaccharides (LPS) of Salmonella minnesota, Salmonella typhimurium and Escherichia coli. The diffraction patterns obtained indicated exclusively lamellar, bilayered arrangements in all samples. The periodicities were found to be in the range 4.5 nm for lipid A to 8.8 nm for Ra-LPS. Upon treatment with water-saturated air, swelling of the lamellar structures was achieved, as indicated by shifts of reflections. The increase in bilayer dimensions normally was about 0.3 nm.X-ray intensities were used for the determination of the inner bilayer structure, i.e. for calculation of the one-dimensional electron-density distribution across the bilayer. For lipid A and several Re-LPS, Rd2-LPS, Rd,-LPS and Rc-LPS samples, a striking coincidence of the electron-density distributions in the lipid-A domain was found, suggesting that in all these structures the lipid-A portion is similarly arranged. For Rb, and Ra-LPS, the lipid-A domain could not be resolved due to the limited number of observed reflections. For other Re-mutant lipopolysaccharide samples, quite different X-ray patterns were obtained. Some samples yielded diffraction patterns indicating a very high state of order in the lipid-A domain, whereas, in others, a significantly reduced order in the lipid-A domain was infered.Comparison of the X-ray data with features of a calculated three-dimensional molecular model of lipopolysaccharide revealed reasonable agreement in molecular dimensions and bilayer structure.Lipopolysaccharides (LPS) are constituents of the outer leaflet of the outer membrane of Gram-negative bacteria [l]. LPS are amphiphilic molecules which differ significantly in structure from simple constituents of other biological membranes such as glycerophospholipids. More precisely, the lipid anchor of LPS, termed lipid A, generally harbours 6-7 saturated fatty acid chains, covalently linked to a diglucosamine head group. Thus, a compact hydrophobic domain is formed which is reported to exhibit a high degree of order [2, 31. To the LPS head group, an oligosaccharide portion is linked. The oligosaccharide structures of different bacteria show structural variations which have been useful in the elucidation of LPS physical behaviour and of the role of distinct core portions in the expression of several physiological reactions induced by LPS [4-91. The general architecture of enterobacterial smooth-strain (S form) LPS is shown in Fig. 1. Structures of rough-mutant (R form) LPS (Ra-LPS to Re-LPS) investigated in this study are defined in Table 1.The physicochemical properties of LPS and its molecular conformation have been studied intensively [lo]. However, up to now, it is not clear if a specific aggregation form [8, 111 of the amphiphilic LPS molecules or a specific confor- mation of a single molecule [12] gives rise to the known endotoxicity of LPS. Unfortunately, a precise X-ray-structure determination of LPS is hampered by the fact that suitabl...

No abstract