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.

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.

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.

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.

Probing the pH-dependent structural features of alpha-KTx12.1, a potassium channel blocker from the scorpion Tityus serrulatus.

Potassium channels are widespread in living cells and are involved in many diseases. The scorpion toxin alpha-KTx(12.1) interacts with various K(+) channels, suggesting its capacity to match diverse channel pores. It is recognized that tissue injuries may affect the pH at toxins site of action, thereby modulating both protein conformation and activity. To better understand its molecular mechanism of action, we studied alpha-KTx(12.1) using pH as a tool to explore its plasticity and NMR in combination with MD calculations to detect it. The toxin solution structure consists of an alpha-helix and a triple-stranded beta-sheet stabilized by four disulfide bridges. The NMR results show, in addition, that His28 possesses an unusually low pK(a) of 5.2. The best set of protein conformers is obtained at pH 4.5, while at pH 7.0, the reduced number of NOEs resulting from a faster hydrogen exchange does not allow to reach a good structural convergence. Nonetheless, MD calculations show that the toxin structure does not vary significantly in that pH range, while conformational changes and modifications of the surface charge distribution occur when His28 is fully protonated. Moreover, essential dynamics analysis reveals variations in the toxin's coherent motions. In conclusion, His28, with its low pK(a) value, provides alpha-KTx(12.1) with the ability to preserve its active conformation over a wide pH interval, thus expanding the range of cellular conditions where the toxin can fully exhibit its activity. Overall, the results further underline the role of histidine as a natural controller of proteins' functionality.

Structure of a synthetic fragment of the LALF protein when bound to lipopolysaccharide.

Peptidic lipopolysaccharide (LPS) antagonists are the subject of intensive research. We report an NMR and modeling study of LALF-14 (GCKPTFRRLKWKYKCG), a synthetic cyclized fragment of the limulus anti-LPS factor (LALF) comprising residues 36-47. In a mixture with LPS we observed the transferred NOE effect and derived the LPS-bound structure of LALF-14. Neither the free nor the LPS-bound peptide displays NOEs indicative of a beta-sheet-like structure that is adopted by the fragment in the full-size protein. However, docking calculations show that the former structure is not a prerequisite for binding of LALF-14 to LPS.

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.

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.

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.

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 alpha-helical and beta-sheet proteins using homology models and/or X-ray structures. The program produced no erroneous 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.

The electron transfer complex between cytochrome c552 and the CuA domain of the Thermus thermophilus ba3 oxidase. A combined NMR and computational approach.

The structural analysis of the redox complex between the soluble cytochrome c552 and the membrane-integral cytochrome ba3 oxidase of Thermus thermophilus is complicated by the transient nature of this protein-protein interaction. Using NMR-based chemical shift perturbation mapping, however, we identified the contact regions between cytochrome c552 and the CuA domain, the fully functional water-soluble fragment of subunit II of the ba3 oxidase. First we determined the complete backbone resonance assignments of both proteins for each redox state. Subsequently, two-dimensional [15N,1H]TROSY spectra recorded for each redox partner both in free and complexed state indicated those surface residues affected by complex formation between the two proteins. This chemical shift analysis performed for both redox states provided a topological description of the contact surface on each partner molecule. Remarkably, very pronounced indirect effects, which were observed on the back side of the heme cleft only in the reduced state, suggested that alterations of the electron distribution in the porphyrin ring due to formation of the protein-protein complex are apparently sensed even beyond the heme propionate groups. The contact residues of each redox partner, as derived from the chemical shift perturbation mapping, were employed for a protein-protein docking calculation that provided a structure ensemble of 10 closely related conformers representing the complex between cytochrome c552 and the CuA domain. Based on these structures, the electron transfer pathway from the heme of cytochrome c552 to the CuA center of the ba3 oxidase has been predicted.

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.

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 alpha-helical (cytochrome c) and beta-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.

Identification of LPS-binding peptide fragment of MD-2, a toll-receptor accessory protein.

Members of the toll-like receptor family are crucial in recognition of microbial pathogens as part of innate immune response. MD-2, an accessory protein to TLR4, present on the extracellular side of the membrane is needed to initiate the signal transduction. We have identified a 15 amino acid region of human MD-2 that contains several features of other lipopolysaccharide (LPS) binding proteins and peptides. In vitro LPS neutralization by this peptide was observed and confirmed by 2D transferred NOESY NMR experiments. NMR experiments have also shown binding of the MD-2 peptide to lipoteichoic acid (LTA) but not to peptidoglycan. Furthermore this peptide inhibited growth of gram-negative and to a lower extent of some gram-positive bacteria. Our results indicate that this region of MD-2 might be responsible for binding of LPS and confirms the role of MD-2 as an accessory protein in LPS signaling bestowing the Toll receptors their specificity.

Modified glycopeptides related to cell wall peptidoglycan: conformational studies by NMR and molecular modelling.

Polymeric peptidoglycans of bacterial cell walls, and smaller glycopeptides derived from them, exhibit versatile biological activities including immunomodulating properties. Peptidoglycan monomer (PGM) was isolated from Brevibacterium divaricatum and novel lipophilic derivatives of PGM bearing either (adamantyl-1-yl)-acetyl or Boc-Tyr substituents (Ad-PGM and BocTyr-PGM respectively) have recently been synthesized. We have obtained full assignments of the 1H and 13C spectra, using 2D NMR techniques, for all three compounds in DMSO solutions. NOESY/ROESY experiments have provided interproton distance restraints that were used in distance geometry modelling calculations to derive conformational preferences for each of these molecules. These data were supplemented with information available from chemical shifts, temperature dependence of amide proton shifts and proton-proton scalar couplings. Analysis of the results suggest that the lipophilic substituents attached to the Dap(3)- epsilon amino group of the parent PGM molecule introduce changes to the conformational preferences of the peptide moiety. In PGM electrostatic interactions between charged end groups apparently promote folded conformations with participation of the long Dap side chain. Derivatives wherein such interactions are suppressed by acylation of the Dap(3)- epsilon amino group are characterized by more extended conformations of the peptide chain. The new synthetic derivatives exhibit biological properties similar to those of the parent PGM. This may indicate that peripheral parts of the peptide chain such as the C-terminal and end groups of the long Dap side chain do not significantly contribute to the binding to receptors or enzymes participating in the biochemical interactions referred to above.

Interaction of cytochrome c with cytochrome c oxidase: an NMR study on two soluble fragments derived from Paracoccus denitrificans.

The functional interactions between the various components of the respiratory chain are relatively short-lived, thus allowing high turnover numbers but at the same time complicating the structural analysis of the complexes. Chemical shift mapping by NMR spectroscopy is a useful tool to investigate such transient contacts, since it can monitor changes in the electron-shielding properties of a protein as the result of temporary contacts with a reaction partner. In this study, we investigated the molecular interaction between two components of the electron-transfer chain from Paracoccus denitrificans: the engineered, water-soluble fragment of cytochrome c(552) and the Cu(A) domain from the cytochrome c oxidase. Comparison of [(15)N,(1)H]-TROSY spectra of the [(15)N]-labeled cytochrome c(552) fragment in the absence and in the presence of the Cu(A) fragment showed chemical shift changes for the backbone amide groups of several, mostly uncharged residues located around the exposed heme edge in cytochrome c(552). The detected contact areas on the cytochrome c(552) surface were comparable under both fully reduced and fully oxidized conditions, suggesting that the respective chemical shift changes represent biologically relevant protein-protein interactions.

A new electron transport mechanism in mitochondrial steroid hydroxylase systems based on structural changes upon the reduction of adrenodoxin.

The adrenal ferredoxin (adrenodoxin, Adx) is an acidic 14.4-kDa [2Fe-2S] ferredoxin that belongs to the vertebrate ferredoxin family. It is involved in the electron transfer from the flavoenzyme NADPH-adrenodoxin-reductase to cytochromes P-450(scc) and P-450(11)(beta). The interaction between the redox partners during electron transport has not yet been fully established. Determining the tertiary structure of an electron-transfer protein may be very helpful in understanding the transport mechanism. In the present work, we report a structural study on the oxidized and reduced forms of bovine adrenodoxin (bAdx) in solution using high-resolution NMR spectroscopy. The protein was produced in Escherichia coli and singly or doubly labeled with (15)N or (13)C/(15)N, respectively. Approximately 70 and 75% of the (15)N, (13)C, and (1)H resonances could be assigned for the reduced and the oxidized bAdx, respectively. The secondary and tertiary structures of the reduced and oxidized states were determined using NOE distance information. (1)H(N)-T(1) relaxation times of certain residues were used to obtain additional distance constraints to the [2Fe-2S] cluster. The results suggest that the solution structure of oxidized Adx is quite similar to the X-ray structure. However, structural changes occur upon reduction of the [2Fe-2S] cluster, as indicated by NMR measurements. It could be shown that these conformational changes, especially in the C-terminal region, cause the dissociation of the Adx dimer upon reduction. A new electron transport mechanism proceeding via a modified shuttle mechanism, with both monomers and dimers acting as electron carriers, is proposed.

Structural analysis of the DNA-binding domain of the Erwinia amylovora RcsB protein and its interaction with the RcsAB box.

The transcriptional regulator RcsB interacts with other coactivators to control the expression of biosynthetic operons in enterobacteria. While in a heterodimer complex with the regulator RcsA the RcsAB box consensus is recognized, DNA binding sites for RcsB without RcsA have also been identified. The conformation of RcsB might therefore be modulated upon interaction with various coactivators, resulting in the recognition of different DNA targets. We report the solution structure of the C-terminal DNA-binding domain of the RcsB protein from Erwinia amylovora spanning amino acid residues 129-215 solved by heteronuclear magnetic resonance (NMR) spectroscopy. The C-terminal domain is composed of four alpha-helices where two central helices form a helix-turn-helix motif similar to the structures of the regulatory proteins GerE, NarL, and TraR. Amino acid residues involved in the RcsA independent DNA binding of RcsB were identified by titration studies with a RcsAB box consensus fragment. Data obtained from NMR spectroscopy together with surface plasmon resonance measurements demonstrate that the RcsAB box is specifically recognized by the RcsAB heterodimer as well as by RcsB alone. However, the binding constant of RcsB alone at target promoters from Escherichia coli, E. amylovora, and Pantoea stewartii was approximately 1 order of magnitude higher compared with that of the RcsAB heterodimer. We present evidence that the obvious role of RcsA is not to alter the DNA binding specificity of RcsB but to stabilize RcsB-DNA complexes.