The cyclic decapeptides polymyxin B (PmB) and E (PmE) (mo-K'TK'-cyclo-[K'K'XLK'K'T]; mo, methyl octanoate; K', diaminobutyric acid; X, D-Phe (PmB) or D-Leu (PmE)) display antimicrobial and lipopolysaccharide (LPS) antagonistic activities. We have investigated the conformational behavior of PmB and PmE in water solution, free and bound to LPS, by homonuclear NMR and molecular modeling methods. The free peptides exist in equilibria of fast exchanging conformations with local preferences for a distorted type II' beta-turn from residues 5-8, and/or a gamma-turn in residue 10. These two motifs are not present in the bound conformation of the peptides. The latter is amphiphilic separating the two hydrophobic residues in the cycle from the positively charged diaminobutyric acid side chains by an envelope-like fold of the cycle. The bound conformation is used for the derivation of a model of the PmB-lipid A complex based on electrostatic interactions and reduction of hydrophobic area. The proposed mode of binding breaks up the supramolecular structure of LPS connected with its toxicity. The model should contribute to the understanding of entropy-driven PmB-lipid A binding at the molecular level and assist the design of inhibitors of endotoxic activity.

Catechins are the main ingredients of green tea extracts and have been shown to possess versatile biological activities, including antimicrobial. We determined that the catechins inhibit bacterial DNA gyrase by binding to the ATP binding site of the gyrase B subunit. In the group of four tested catechins, epigallocatechin gallate (EGCG) had the highest activity, followed by epicatechin gallate (ECG) and epigallocatechin (EGC). Specific binding to the N-terminal 24 kDa fragment of gyrase B was determined by fluorescence spectroscopy and confirmed using heteronuclear two-dimensional NMR spectroscopy of the EGCG-15N-labeled gyrase B fragment complex. Protein residues affected by binding to EGCG were identified through chemical shift perturbation. Molecular docking calculations suggest that the benzopyran ring of EGCG penetrates deeply into the active site while the galloyl moiety anchors it to the cleft through interactions with its hydroxyl groups, which explains the higher activity of EGCG and ECG.

Curcumin is the main constituent of the spice turmeric, used in diet and in traditional medicine, particularly across the Indian subcontinent. Anti-inflammatory activity and inhibition of LPS signaling are some of its many activities. We show that curcumin binds at submicromolar affinity to the myeloid differentiation protein 2 (MD-2), which is the LPS-binding component of the endotoxin surface receptor complex MD-2/TLR4. Fluorescence emission of curcumin increases with an absorbance maximum shift toward the blue upon the addition of MD-2, indicating the transfer of curcumin into the hydrophobic environment. Curcumin does not form a covalent bond to the free thiol group of MD-2, and C133F mutant retains the binding and inhibition by curcumin. The binding site for curcumin overlaps with the binding site for LPS. This results in the inhibition of MyD88-dependent and -independent signaling pathways of LPS signaling through TLR4, indicating that MD-2 is one of the important targets of curcumin in its suppression of the innate immune response to bacterial infection. This finding, in addition to the correlation between the dietary use of curcumin and low incidence of gastric cancer in India, may have important implications for treatment and epidemiology of chronic inflammatory diseases caused by bacterial infection.

Gram-negative bacterial endotoxin (i.e. lipopolysaccharide (LPS)) is one of the most potent stimulants of the innate immune system, recognized by the TLR4⅐MD-2 complex. Direct binding to MD-2 of LPS and LPS analogues that act as TLR4 agonists or antagonists is well established, but the role of MD-2 and TLR4 in receptor activation is much less clear. We have identified residues within the hairpin of MD-2 between strands five and six that, although not contacting acyl chains of tetraacylated lipid IVa (a TLR4 antagonist), influence activation of TLR4 by hexaacylated lipid A. We show that hydrophobic residues at positions 82, 85, and 87 of MD-2 are essential both for transfer of endotoxin from CD14 to monomeric MD-2 and for TLR4 activation. We also identified a pair of conserved hydrophobic residues (Phe-440 and Phe-463) in leucine-rich repeats 16 and 17 of the TLR4 ectodomain, which are essential for activation of TLR4 by LPS. F440A or F463A mutants of TLR4 were inactive, whereas the F440W mutant retained full activity. Charge reversal of neighboring cationic groups in the TLR4 ectodomain (Lys-388 and Lys-435), in contrast, did not affect cell activation. Our mutagenesis studies are consistent with a molecular model in which Val-82, Met-85, and Leu-87 in MD-2 and distal portions of a secondary acyl chain of hexaacylated lipid A that do not fit into the hydrophobic binding pocket of MD-2 form a hydrophobic surface that interacts with Phe-440 and Phe-463 on a neighboring TLR4⅐MD-2⅐LPS complex, driving TLR4 activation. Bacterial lipopolysaccharide (LPS)3 is recognized by the innate immune system of vertebrates via an elaborate mechanism involving the membrane receptor TLR4 (1, 2). The extracellular (or cell surface) proteins LPS-binding protein and CD14 promote extraction and transfer of individual molecules of LPS from the Gram-negative bacterial outer membrane to MD-2, either secreted monomeric soluble (s)MD-2 or MD-2 bound with high affinity to the ectodomain of TLR4 (3-7). In contrast to other Toll-like receptors, TLR4 requires an additional molecule, MD-2, for ligand recognition (8). In contrast to MD-2, there has been no evidence of direct binding of LPS to TLR4 (9, 10). Although LPS, and particularly the lipid A portion of LPS, is generally conserved among Gram-negative bacteria, there are many variables in LPS structure that affect TLR4 activation. Most important is the acylation pattern of the lipid A moiety, which represents the minimal segment of LPS that can trigger activation of TLR4 (11). Comparison of crystal structures of MD-2 with and without bound tetraacylated lipid IVa indicates no significant alteration of the protein fold in the absence or presence of bound ligand (12). It has been proposed that both LPS and MD-2 are key to the different effects of tetraversus hexaacylated LPS on TLR4 (8,13,14). Lipid IVa complexed to murine MD-2 has weak agonist effects on murine TLR4 but acts as a receptor antagonist in the same complex containing human MD-2. Hexaacylated endotoxins complexed to human or murin...

Prion diseases are fatal transmissible neurodegenerative diseases affecting many mammalian species. The normal prion protein (PrP) converts into a pathological aggregated form, PrP Sc , which is enriched in the ␤-sheet structure. Although the high resolution structure of the normal PrP was determined, the structure of the converted form of PrP remains inaccessible to high resolution techniques. To map the PrP conversion process we introduced disulfide bridges into different positions within the globular domain of PrP, tethering selected secondary structure elements. The majority of tethered PrP mutants exhibited increased thermodynamic stability, nevertheless, they converted efficiently. Only the disulfides that tether subdomain B1-H1-B2 to subdomain H2-H3 prevented PrP conversion in vitro and in prion-infected cell cultures. Reduction of disulfides recovered the ability of these mutants to convert, demonstrating that the separation of subdomains is an essential step in conversion. Formation of disulfide-linked proteinase K-resistant dimers in fibrils composed of a pair of single cysteine mutants supports the model based on domain-swapped dimers as the building blocks of prion fibrils. In contrast to previously proposed structural models of PrP Sc suggesting conversion of large secondary structural segments, we provide evidence for the conservation of secondary structural elements of the globular domain upon PrP conversion. Previous studies already showed that dimerization is the rate-limiting step in PrP conversion. We show that separation and swapping of subdomains of the globular domain is necessary for conversion. Therefore, we propose that the domain-swapped dimer of PrP precedes amyloid formation and represents a potential target for therapeutic intervention.Prion diseases, also called transmissible spongiform encephalopathies, are neurodegenerative diseases affecting a variety of mammalian species from mink to cow, with human being no exception. In these diseases, cellular prion protein (PrP C ) 5 converts into the aggregated form PrP Sc , which is the main component of the infectious agents, the prions (1). PrP C is a glycosylphosphatidylinositol-anchored protein found on the membranes of neurons and many other cells (2). The N-terminal half of the protein, which is devoid of a defined tertiary structure (3), is followed by a C-terminal globular domain composed of three ␣-helices (H1, H2, H3) and a short antiparallel ␤-sheet (composed of two strands, B1 and B2) (Fig. 1A) (4). High resolution structures of the C-terminal domain of PrP from different species revealed the conservation of the protein fold (5). In contrast to the availability of structural information on PrP C , the characteristics of PrP Sc make it inaccessible to high-resolution structural techniques (x-ray crystallography and high resolution NMR). PrP Sc is characterized by the increased content of the ␤-secondary structure in comparison to PrP C (6 -8). Epitope accessibility (9 -12), deuterium exchange (13-15), limited proteolysis and mas...