Globular domain of the prion protein needs to be unlocked by domain swapping to support prion protein conversion.

Prion diseases are fatal transmissible neurodegenerative diseases affecting many mammalian species. The normal prion protein (PrP) converts into a pathological aggregated form, PrPSc, which is enriched in the ß-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 PrPSc 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.

Taxanes inhibit human TLR4 signaling by binding to MD-2.

LPS is the primary ligand of Toll-like receptor 4, activating it through binding to its accessory protein MD-2. Murine but not human cells expressing MD-2/TLR4 are also activated by paclitaxel. Paclitaxel binds to human MD-2. The binding site of paclitaxel overlaps with the binding site of bis-ANS and LPS, which results in the ability of taxanes to inhibit LPS signaling in the system with human receptors. Circular dichroic spectra of human MD-2 indicated differences in the chemical environment in the presence of paclitaxel and docetaxel. Molecular docking identified the interacting residues of MD-2 and suggests that hydrophobic interactions govern the binding, while the C-3'N group where the paclitaxel and docetaxel differ is exposed on the surface of MD-2.

Structural aspects of peptides with immunomodulating activity.

The main function of the innate immune system from insects to mammals is to detect the presence of and act against invading microorganisms by recognizing their unique molecular signatures, most importantly, components of bacterial cell walls. A large number of peptides and derivatives, both synthetic and of natural origin, are known to influence immune responses in mammals by interacting with the conserved microbial structures, making the former attractive targets for drug development. This review focuses on structural aspects of the immunomodulating peptides and their receptors, including primary constitution, stereochemistry, conformation, binding and hydrophobic properties.

MD-2 as the target of curcumin in the inhibition of response to LPS.

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.

Green tea catechins inhibit bacterial DNA gyrase by interaction with its ATP binding site.

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.

Structure of a synthetic fragment of the lipopolysaccharide (LPS) binding protein when bound to LPS and design of a peptidic LPS inhibitor.

Peptidic lipopolysaccharide (LPS) antagonists are the subject of intensive research. We report an NMR and modeling study of LBP-14 (RVQGRWKVRASFFK), a synthetic fragment of the LPS binding protein (LBP). In a mixture with LPS we observed the transferred nuclear Overhauser effect and determined the LPS-bound structure of LBP-14 that was used for docking calculations to LPS. The derived complex was used to design a peptide that displayed more than 50% increase in LPS inhibition in vitro.

Structural origin of endotoxin neutralization and antimicrobial activity of a lactoferrin-based peptide.

Treatment of Gram-negative bacterial infections with antimicrobial agents can cause release of the endotoxin lipopolysaccharide (LPS), the potent initiator of sepsis, which is the major cause of mortality in intensive care units worldwide. Structural information on peptides bound to LPS can lead to the development of more effective endotoxin neutralizers. Short linear antimicrobial and endotoxin-neutralizing peptide LF11, based on the human lactoferrin, binds to LPS, inducing a peptide fold with a "T-shaped" arrangement of a hydrophobic core and two clusters of basic residues that match the distance between the two phosphate groups of LPS. Side chain arrangement of LF11 bound to LPS extends the previously proposed LPS binding pattern, emphasizing the importance of both electrostatic and hydrophobic interactions in a defined geometric arrangement. In anionic micelles, the LF11 forms amphipathic conformation with a smaller hydrophobic core than in LPS, whereas in zwitterionic micelles, the structure is even less defined. Protection of tryptophan fluorescence quenching in the order SDS>LPS>DPC and hydrogen exchange protection indicates the decreasing extent of insertion of the N terminus and potential role of peptide plasticity in differentiation between bacterial and eukaryotic membranes.

The search for molecular determinants of LPS inhibition by proteins and peptides.

Lipo-poly-saccharide (LPS) induced Gram-negative sepsis and septic shock remain lethal in up to 60 % of cases, and LPS antagonists that neutralize its endotoxic action are the subject of intensive research. The molecular motifs of specific binding of LPS by antiendotoxin proteins and peptides may lead to an understanding of LPS action at the atomic level and provide clues for the development of new immunomodulatory compounds for use as therapy in the treatment of Gram-negative bacterial sepsis. The interaction of LPS with its cognate binding proteins has been structurally elucidated in the single case of the X-ray crystallographic structure of LPS in complex with the integral outer membrane protein FhuA from E. coli K-12 (Ferguson et al., Science 1999, 282, 2215). This structure and other known structures of LPS binding proteins have been used to propose a common binding motif of LPS to proteins. Another independent source of structural information are solution structures of peptides in complex with LPS that can be determined using the transferred NOE effect. The molecular mechanisms of biological activity of bacterial endotoxins can additionally be probed by theoretical means. The growing structural knowledge is opening pathways to the design of peptides or peptidomimetics with improved antiendotoxin properties.

Solution structure and stability of the full-length excisionase from bacteriophage HK022.

Heteronuclear high-resolution NMR spectroscopy was employed to determine the solution structure of the excisionase protein (Xis) from the lambda-like bacteriophage HK022 and to study its sequence-specific DNA interaction. As wild-type Xis was previously characterized as a generally unstable protein, a biologically active HK022 Xis mutant with a single amino acid substitution Cys28-->Ser was used in this work. This substitution has been shown to diminish the irreversibility of Xis denaturation and subsequent degradation, but does not affect the structural or thermodynamic properties of the protein, as evidenced by NMR and differential scanning calorimetry. The solution structure of HK022 Xis forms a compact, highly ordered protein core with two well-defined alpha-helices (residues 5-11 and 18-27) and five beta-strands (residues 2-4, 30-31, 35-36, 41-44 and 48-49). These data correlate well with 1H2O-2H2O exchange experiments and imply a different organization of the HK022 Xis secondary structure elements in comparison with the previously determined structure of the bacteriophage lambda excisionase. Superposition of both Xis structures indicates a better correspondence of the full-length HK022 Xis to the typical 'winged-helix' DNA-binding motif, as found, for example, in the DNA-binding domain of the Mu-phage repressor. Residues 51-72, which were not resolved in the lambda Xis, do not show any regular structure in HK022 Xis and thus appear to be completely disordered in solution. The resonance assignments have shown, however, that an unusual connectivity exists between residues Asn66 and Gly67 owing to asparagine-isoaspartyl isomerization. Such an isomerization has been previously observed and characterized only in eukaryotic proteins.