A unique peptide-based pharmacophore identifies an inhibitory compound against the A-subunit of Shiga toxin.

Shiga toxin (Stx), a major virulence factor of enterohemorrhagic Escherichia coli (EHEC), can cause fatal systemic complications. Recently, we identified a potent inhibitory peptide that binds to the catalytic A-subunit of Stx. Here, using biochemical structural analysis and X-ray crystallography, we determined a minimal essential peptide motif that occupies the catalytic cavity and is required for binding to the A-subunit of Stx2a, a highly virulent Stx subtype. Molecular dynamics simulations also identified the same motif and allowed determination of a unique pharmacophore for A-subunit binding. Notably, a series of synthetic peptides containing the motif efficiently inhibit Stx2a. In addition, pharmacophore screening and subsequent docking simulations ultimately identified nine Stx2a-interacting molecules out of a chemical compound database consisting of over 7,400,000 molecules. Critically, one of these molecules markedly inhibits Stx2a both in vitro and in vivo, clearly demonstrating the significance of the pharmacophore for identifying therapeutic agents against EHEC infection.

Identification of a peptide motif that potently inhibits two functionally distinct subunits of Shiga toxin.

Shiga toxin (Stx) is a major virulence factor of enterohemorrhagic Escherichia coli, which causes fatal systemic complications. Here, we identified a tetravalent peptide that inhibited Stx by targeting its receptor-binding, B-subunit pentamer through a multivalent interaction. A monomeric peptide with the same motif, however, did not bind to the B-subunit pentamer. Instead, the monomer inhibited cytotoxicity with remarkable potency by binding to the catalytic A-subunit. An X-ray crystal structure analysis to 1.6 Å resolution revealed that the monomeric peptide fully occupied the catalytic cavity, interacting with Glu167 and Arg170, both of which are essential for catalytic activity. Thus, the peptide motif demonstrated potent inhibition of two functionally distinct subunits of Stx.

Adsorption Behaviors of Mixed Monolayers of n-Alkanes at the Liquid-Solid Interface.

To understand the self-assembly of monolayers at the liquid-solid interface, a thermodynamic model, which describes the contributions of the molecular interactions, is essential. We present an adapted Zimm-Bragg model of the cooperativity transitions for determining the Gibbs free energy for self-assembly at the liquid-solid interface. Scanning tunneling microscopy was used to observe the monolayers formed on graphite from phenyloctane solutions of binary mixtures of n-hexacosane (C26H54) and n-tetratriacontane (C34H70). This revealed that the sharp transition in the monolayers from the full surface coverage of the long-chain alkane, which is adsorbed preferentially, to the full coverage of the short-chain alkane is a function of the mixture composition. The model allows for the estimation of the free-energy changes associated with the difference in the alkyl chain length and the interface between the two different alkane regions in the monolayers. It is also suitable for understanding more complex systems that exhibit intermolecular interactions.

Self-assembled monolayers of cholesterol and cholesteryl esters on graphite.

The molecular arrangements of self-assembled monolayers (SAMs) of cholesterol, cholesteryl laurate, and cholesteryl stearate adsorbed on a graphite surface were studied using scanning tunneling microscopy (STM) at the liquid-solid interface. The STM images of the SAMs showed two-dimensional periodic arrays of bright regions that corresponded to the sterol rings. However, individual sterol rings could not be observed in the bright regions in the STM images of the cholesterol monolayers. Nevertheless, by comparing the STM images and the crystallographic data, it is concluded that the cholesterol molecules are arranged in pairs oriented head-to-head owing to the hydrogen bonds between the hydroxyl groups. These dimers, in turn, are oriented parallel to each other, owing to the interactions between the sterol rings. The STM images of cholesteryl ester monolayers had molecular resolution and showed pairs of cholesteryl ester molecules oriented in an antiparallel manner, with their fatty acid chains located in the central regions. Furthermore, the fatty acid chains of cholesteryl stearate were observed to be oriented in the (1120) zigzag direction of the graphite lattice, whereas those of cholesteryl laurate were oriented in the (1010) armchair direction. These observations reveal that the interactions between the fatty acid chains affect the structure of the SAMs. The molecular arrangements also depend on the lengths of the fatty acid chains of the cholesterol esters and hence on the interactions between the alkyl chains and the graphite surface. The self-assembly at the liquid-solid interface is therefore controlled by the interactions between sterol rings, between alkyl chains, and between alkyl chains and the substrate.

Cooperative rotation of alkylated sulfide molecules at the liquid-graphite interface.

Self-assembled monolayers of hexadecyl sulfide adsorbed on a graphite surface were studied using scanning tunneling microscopy (STM). STM images of the molecule indicate a bright spot and two relatively dim thin bands that correspond to the sulfur atom positioned at the center of the molecule and two alkyl chains that extend linearly from the sulfur atom, respectively. The contrast of the bands changes reversibly between the zigzag and aligned bright spots, which correspond to the zigzag methylene units of the alkyl chains; accordingly, the methylene units alternate between parallel and perpendicular orientations to the surface, respectively, on a time scale of minutes. The variation in contrast indicates cooperative rotation along the long axis of the molecules in the monolayer at the liquid-graphite interface. The reversibility of the contrast suggests that the solvent influences the difference in the free energies of the parallel and perpendicular configurations of the molecules with respect to the graphite surface.

Chemical force microscopy using functionalized ZnO whisker probe tips.

We have developed an atomic force microscope-based technique utilizing a zinc oxide whisker crystal as a probe tip. This technique was used for measuring interactions between the chemically modified tip and oxidized silicon substrates as a function of solution pH. Surfaces terminating in amine and methyl functional groups were prepared by covalently modifying the silicon substrate with self-assembled monolayers of (3-amino-propyl)triethoxysilane and n-butyltrichlorosilane, respectively. Patterned samples prepared by microcontact printing consisted of amine-terminated square regions surrounded by a methyl-terminated background. The contrast in lateral force images of the patterned samples obtained with amine-functionalized tips was seen to strongly depend on solution pH. High friction was observed between the attractive and strongly interacting functional groups in the methyl-terminated regions at low pH values and the amine-terminated regions at intermediate pH values. Low friction was observed between repulsive and weakly interacting functional groups. In addition, interacting force measurements on approach showed long-range attractive forces between the amine-terminated surfaces at intermediate pH values. Adhesive force measurements showed a dependency on the state of ionization of the amine groups, which was controlled by varying the pH. The pKa value estimated from the measurements was shifted to a lower pH than that of primary amine groups in solution. These results show that functionalized ZnO whisker probe tips have great potential for chemically sensitive imaging.

Collapse in binary phospholipid monolayers at the air/water interface.

The behavior in the stochastic collapse of lipid monolayers of a 7:3 mixture of DPPC (dipalmitoylphosphatidylcholine) and POPG (palmitoyloleoylphosphatidylglycerol), a model system of pulmonary surfactant assembly, was studied using Langmuir isotherms, fluorescence microscopy, and atomic force microscopy. The monolayers at the air-water interface appeared to be phase separated under compression and retained the continuous liquid-expanded phase network surrounding islands of condensed phase even at a surface pressure approaching 70 mN/m. When the two-dimensional monolayers were compressed beyond the equilibrium surface pressure, they collapsed and assumed a three-dimensional formation. Collapse events involved folding of the monolayer on a micron scale, and each event produced a macroscopic jerk of the layer. The distribution of waiting times between events was estimated to be an exponential function, indicating that the events were independent. Folded regions coexisted with the flat monolayer, remained attached to the interface, and reversibly reincorporated into the monolayer upon expansion.