Primož Pristovšek

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.

Essential Roles of Hydrophobic Residues in Both MD-2 and Toll-like Receptor 4 in Activation by Endotoxin

Gram-negative bacterial endotoxin (i.e. lipopolysaccharide (LPS)) is one of the most potent stimulants of the innate immune system, recognized by the TLR4·MD-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 TLR4·MD-2·LPS complex, driving TLR4 activation.

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.

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 β-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.

Semiautomatic sequence-specific assignment of proteins based on the tertiary structure—The program st2nmr

The sequence‐specific assignment of resonances is still the most time‐consuming procedure that is necessary as the first step in high‐resolution NMR studies of proteins. In many cases a reliable three‐dimensional (3D) structure of the protein is available, for example, from X‐ray spectroscopy or homology modeling. Here we introduce the st2nmr program that uses the 3D structure and Nuclear Overhauser Effect spectroscopy (NOESY) peak list(s) to evaluate and optimize trial sequence‐specific assignments of spin systems derived from correlation spectra to residues of the protein. A distance‐dependent target function that scores trial assignments based on the presence of expected NOESY crosspeaks is optimized in a Monte Carlo fashion. The performance of the program st2nmr is tested on real NMR data of an α‐helical (cytochrome c) and β‐sheet (lipocalin) protein using homology models and/or X‐ray structures; it succeeded in completely reproducing the correct sequence‐specific assignments in most cases using 2D and/or 15N/13C Nuclear Overhauser Effect (NOE) data. Additionally to amino acid residues the program can also handle ligands that are bound to the protein, such as heme, and can be used as a complementary tool to fully automated assignment procedures.

Solution structure and dynamics of the functional domain of Paracoccus denitrificans cytochrome c(552) in both redox states.

A soluble and fully functional 10.5 kDa fragment of the 18.2 kDa membrane-bound cytochrome c(552) from Paracoccus denitrificans has been heterologously expressed and (13)C/(15)N-labeled to study the structural features of this protein in both redox states. Well-resolved solution structures of both the reduced and oxidized states have been determined using high-resolution heteronuclear NMR. The overall protein topology consists of two long terminal helices and three shorter helices surrounding the heme moiety. No significant redox-induced structural differences have been observed. (15)N relaxation rates and heteronuclear NOE values were determined at 500 and 600 MHz. Several residues located around the heme moiety display increased backbone mobility in both oxidation states, while helices I, III, and V as well as the two concatenated beta-turns between Leu30 and Arg36 apparently form a less flexible domain within the protein structure. Major redox-state-dependent differences of the internal backbone mobility on the picosecond-nanosecond time scale were not evident. Hydrogen exchange experiments demonstrated that the slow-exchanging amide proton resonances mainly belong to the helices and beta-turns, corresponding to the regions with high order parameters in the dynamics data. Despite this correlation, the backbone amide protons of the oxidized cytochrome c(552) exchange considerably faster with the solvent compared to the reduced protein. Using both differential scanning calorimetry as well as temperature-dependent NMR spectroscopy, a significant difference in the thermostabilities of the two redox states has been observed, with transition temperatures of 349.9 K (76.8 degrees C) for reduced and 307.5 K (34.4 degrees C) for oxidized cytochrome c(552). These results suggest a clearly distinct backbone stability between the two oxidation states.

Stereospecific assignments of protein NMR resonances based on the tertiary structure and 2D/3D NOE data

In many cases of protein structure determination by NMR a high‐quality structure is required. An important contribution to structural precision is stereospecific assignment of magnetically nonequivalent prochiral methylene and methyl groups, eliminating the need for introducing pseudoatoms and pseudoatom corrections in distance restraint lists. Here, we introduce the stereospecific assignment program that uses the resonance assignment, a preliminary 3D structure and 2D and/or 3D nuclear Overhauser effect spectroscopy peak lists for stereospecific assignment. For each prochiral group the algorithm automatically calculates a score for the two different stereospecific assignment possibilities, taking into account the presence and intensity of the nuclear Overhauser effect (NOE) peaks that are expected from the local environment of each prochiral group (i.e., the close neighbors). The performance of the algorithm has been tested and used on NMR data of α‐helical and β‐sheet proteins using homology models and/or X‐ray structures. The program produced no erroneus stereospecific assignments provided the NOEs were carefully picked and the 3D model was sufficiently accurate. The set of NOE distance restraints produced by nmr2st using the results of the SSA module was superior in generating good‐quality ensembles of NMR structures (low deviations from upper limits in conjunction with low root‐mean‐square‐deviation values) in the first round of structure calculations. The program uses a novel approach that employs the entire 3D structure of the protein to obtain stereospecific assignment; it can be used to speed up the NMR structure refinement and to increase the quality of the final NMR ensemble even when no scalar or residual dipolar coupling information is available.

Interaction of the HIV-1 gp120 Viral Protein V3 Loop with Bacterial Lipopolysaccharide A PATTERN RECOGNITION INHIBITION

HIV-1 represents an elusive target for therapeutic compounds due to its high rate of mutation. Targeting structural patterns instead of a constantly changing specific three-dimensional structure may represent an approach that is less sensitive to viral mutations. The V3 loop of gp120 of HIV-1, which is responsible for binding of viral gp120 to CCR5 or CXCR4 coreceptors, has already been identified as an effective target for the inhibition of viral entry. The peptide derived from the V3 loop of gp120 specifically interacts with the lipid A moiety of LPS, as does the full gp120 protein. NMR analysis of V3 in complex with LPS shows formation of an amphipathic turn. The interaction between LPS and V3 relies on the structural pattern, comprising a combination of hydrophobic and charge interactions, similar to the interaction between antimicrobial peptides and LPS. LPS inhibited binding of gp120 to the surface of target T cells. Nonendotoxic LPS antagonists inhibited viral infection, demonstrating the possibility for the development of an inhibitor of HIV-1 attachment to T cells based on the recognition of a conserved structural pattern.