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Funnel metadynamics is a kind of computational simulation used to enhance the sampling of protein-ligand binding events in solution. By characterization of the binding interaction events, an estimated absolute binding free energy can be calculated. Nuclear magnetic resonance and funnel metadynamics were used to evaluate the binding of pyrocatechol derivatives (catechol, 4-methylcatechol, and 4-tert-butylcatechol) to human peroxiredoxin 5. Human peroxiredoxins are peroxidases involved in cellular peroxide homeostasis. Recently, overexpressed or suppressed peroxiredoxin levels have been linked to various diseases. Here, the catechol derivatives were found to be inhibitors against human peroxiredoxin 5 through a partial mixed type noncompetitive mechanism. Funnel metadynamics provided a microscopic model for interpreting the inhibition mechanism. Correlations were observed between the inhibition constants and the absolute binding free energy. Overall, this study showcases the fact that funnel metadynamics simulations can be employed as a preliminary approach to gain an in-depth understanding of potential enzyme inhibitors.

One of the intrinsic properties of proteins is their capacity to interact selectively with other molecules in their environment, inducing many chemical equilibria each differentiated by the mutual affinities of the components. A comprehensive understanding of these molecular binding processes at atomistic resolution requires formally the complete description of the system dynamics and statistics at the relevant time scales. While solution NMR observables are averaged over different time scales, from picosecond to second, recent new molecular dynamics protocols accelerated considerably the simulation time of realistic model systems. Based on known ligands recently discovered either by crystallography or NMR for the human peroxiredoxin 5, their affinities were for the first time accurately evaluated at atomistic resolution comparing absolute binding free-energy estimated by funnel-metadynamics simulations and solution NMR experiments. In particular, free-energy calculations are demonstrated to discriminate two closely related ligands as pyrocatechol and 4-methylpyrocathecol separated just by 1 kcal/mol in aqueous solution. The results provide a new experimental and theoretical basis for the estimation of ligand-protein affinities.

To date, the signal transducing adaptor molecule 2 (STAM2) was shown to harbour two ubiquitin binding domains (UBDs) known as the VHS and UIM domains, while the SH3 domain of STAM2 was reported to interact with deubiquitinating enzymes (DUBs) like UBPY and AMSH. In the present study, NMR evidences the interaction of the STAM2 SH3 domain with ubiquitin, demonstrating that SH3 constitutes the third UBD of STAM2. Furthermore, we show that a UBPY-derived peptide can outcompete ubiquitin for SH3 binding and vice versa. These results suggest that the SH3 domain of STAM2 plays versatile roles in the context of ubiquitin mediated receptor sorting., (Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Muscle creatine kinase (MCK; EC2.7.3.2) is a 86 kDa homodimer that belongs to the family of guanidino kinases. MCK has been intensively studied for several decades, but it is still not known why it is a dimer because this quaternary structure does not translate into obvious structural or functional advantages over the homologous monomeric arginine kinase. In particular, it remains to be demonstrated whether MCK subunits are independent. Here, we describe NMR chemical-shift perturbation and relaxation experiments designed to study the active site 320s flexible loop of this enzyme. The analysis was performed with the enzyme in its ligand-free and MgADP-complexed forms, as well as with the transition-state analogue abortive complex (MCK-Mg-ADP-creatine-nitrate ion). Our data indicate that each subunit can bind substrates independently., (© 2012 The Authors Journal compilation © 2012 FEBS.)

As the upstream component of the ESCRT (endosomal sorting complexes required for transport) machinery, the ESCRT-0 complex is responsible for directing ubiquitinated membrane proteins to the multivesicular body pathway. ESCRT-0 is formed by two subunits known as Hrs (hepatocyte growth factor-regulated substrate) and STAM (signal transducing adaptor molecule), both of which harbor multiple ubiquitin-binding domains (UBDs). In particular, STAM2 possesses two UBDs, the VHS (Vps27/Hrs/Stam) and UIM (ubiquitin interacting motif) domains, connected by a 20-amino acid flexible linker. In the present study, we report the interactions of the UIM domain and VHS-UIM construct of STAM2 with monoubiquitin (Ub), Lys(48)- and Lys(63)-linked diubiquitins. Our results demonstrate that the UIM domain alone binds monoubiquitin, Lys(48)- and Lys(63)-linked diubiquitins with the same affinity and in the same binding mode. Interestingly, binding of VHS-UIM to Lys(63)-linked diubiquitin is not only avid, but also cooperative. We also show that the distal domain of Lys(63)-linked diubiquitin stabilizes the helical structure of the UIM domain and that the corresponding complex adopts a specific structural organization responsible for its greater affinity. In contrast, binding of VHS-UIM to Lys(48)-linked diubiquitin and monoubiquitin is not cooperative and does not show any avidity. These results may explain the better sorting efficiency of some cargoes polyubiquitinated with Lys(63)-linked chains over monoubiquitinated cargoes or those tagged with Lys(48)-linked chains.

Fragment-based drug discovery has become a powerful method for the generation of drug leads against therapeutic targets. Beyond the identification of novel and effective starting points for drug design, fragments have emerged as reliable tools for assessing protein druggability and identifying protein hot spots. Here, we have examined fragments resulting from the deconstruction of known inhibitors from the glycogen phosphorylase enzyme, a therapeutic target against type 2 diabetes, with two motivations. First, we have analyzed the fragment binding to the multiple binding sites of the glycogen phosphorylase, and then we have investigated the use of fragments to study allosteric enzymes. The work we report illustrates the power of fragmentlike ligands not only for probing the various binding pockets of proteins, but also for uncovering cooperativity between these various binding sites.

Programmed cell death (PCD) has a key role in defence and development of all multicellular organisms. In plants, there is a large gap in our knowledge of the molecular machinery involved at the various stages of PCD, especially the early steps. Here, we identify kiss of death (KOD) encoding a 25-amino-acid peptide that activates a PCD pathway in Arabidopsis thaliana. Two mutant alleles of KOD exhibited a reduced PCD of the suspensor, a single file of cells that support embryo development, and a reduced PCD of root hairs after a 55°C heat shock. KOD expression was found to be inducible by biotic and abiotic stresses. Furthermore, KOD expression was sufficient to cause death in leaves or seedlings and to activate caspase-like activities. In addition, KOD-induced PCD required light in leaves and was repressed by the PCD-suppressor genes AtBax inhibitor 1 and p35. KOD expression resulted in depolarization of the mitochondrial membrane, placing KOD above mitochondria dysfunction, an early step in plant PCD. A KOD∷GFP fusion, however, localized in the cytosol of cells and not mitochondria.

The VHS domain of the Stam2 protein is a ubiquitin binding domain involved in the recognition of ubiquitinated proteins committed to lysosomal degradation. Among all VHS domains, the VHS domain of Stam proteins is the strongest binder to monoubiqiuitin and exhibits preferences for K63-linked chains. In the present paper, we report the solution NMR structure of the Stam2-VHS domain in complex with monoubiquitin by means of chemical shift perturbations, spin relaxation, and paramagnetic relaxation enhancements. We also characterize the interaction of Stam2-VHS with K48- and K63-linked diubiquitin chains and report the first evidence that VHS binds differently to these two chains. Our data reveal that VHS enters the hydrophobic pocket of K48-linked diubiquitin and binds the two ubiquitin subunits with different affinities. In contrast, VHS interacts with K63-linked diubiquitin in a mode similar to its interaction with monoubiquitin. We also suggest possible structural models for both K48- and K63-linked diubiquitin in interaction with VHS. Our results, which demonstrate a different mode of binding of VHS for K48- and K63-linked diubiquitin, may explain the preference of VHS for K63- over K48-linked diubiquitin chains and monoubiquitin.

Photolysable caged ligands are used to investigate protein function and activity. Here, we investigate the binding properties of caged nucleotides and their photo released products to well established but evolutionary and structurally unrelated nucleotide-binding proteins, rabbit muscle creatine kinase (RMCK) and human annexin A6 (hAnxA6), using saturation transfer difference NMR spectroscopy. We detect the binding of the caged nucleotides and discuss the general implications on interpreting data collected with photolysable caged ligands using different techniques. Strategies to avoid non-specific binding of caged compound to certain proteins are also suggested., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Fragment-based drug design consists of identifying low-molecular weight compounds that weakly bind to a target macromolecule and will then be modified or linked to yield potent inhibitors. The specificity of these low-complexity and low-affinity molecules has rarely been discussed in the literature. To address this question, NMR spectroscopy was used to investigate the interactions of 150 fragments with five proteins: three proteins from the Bcl-2 family (Bcl-x(L), Bcl-w, and Mcl-1), human peroxiredoxin 5, for which very few ligands have been reported, and human serum albumin, which is known to bind a large number of ligands. Our results show that the fragments are rather versatile binders and able to identify binding hot spots in very different targets. Despite the different hit rates observed related to the druggability of the proteins, two scaffolds appear as preferred binders for all proteins. Low specificity was observed between homologous proteins or unrelated poorly druggable proteins, while higher specificity could be achieved with highly druggable targets.

Fragment-based drug design consists of screening low-molecular-weight compounds in order to identify low-affinity ligands that are then modified or linked to yield potent inhibitors. The method thus attempts to build bioactive molecules in a modular way and relies on the hypothesis that the fragment binding mode will be conserved upon elaboration of the active molecule. If the inverse process is considered, do the fragments resulting from the deconstruction of high-affinity inhibitors recapitulate their binding mode in the large molecule? Few studies deal with this issue. Here, we report the analysis of 22 fragments resulting from the dissection of 9 inhibitors of the antiapoptotic protein Bcl-x(L). To determine if the fragments retained affinity toward the protein and identify their binding site, ligand-observed and protein-observed NMR experiments were used. The analysis of the fragments behavior illustrates the complexity of low-affinity protein-ligand interactions involved in the fragment-based construction of bioactive molecules.

The search for protein ligands is a crucial step in the inhibitor design process. Fragment screening represents an interesting method to rapidly find lead molecules, as it enables the exploration of a larger portion of the chemical space with a smaller number of compounds as compared to screening based on drug-sized molecules. Moreover, fragment screening usually leads to hit molecules that form few but optimal interactions with the target, thus displaying high ligand efficiencies. Here we report the screening of a homemade library composed of 200 highly diverse fragments against the human Peroxiredoxin 5 protein. Peroxiredoxins compose a family of peroxidases that share the ability to reduce peroxides through a conserved cysteine. The three-dimensional structures of these enzymes ubiquitously found throughout evolution have been extensively studied, however, their biological functions are still not well understood and to date few inhibitors have been discovered against these enzymes. Six fragments from the library were shown to bind to the Peroxiredoxin 5 active site and ligand-induced chemical shift changes were used to drive the docking of these small molecules into the protein structure. The orientation of the fragments in the binding pocket was confirmed by the study of fragment homologues, highlighting the role of hydroxyl functions that hang the ligands to the Peroxiredoxin 5 protein. Among the hit fragments, the small catechol molecule was shown to significantly inhibit Peroxiredoxin 5 activity in a thioredoxin peroxidase assay. This study reports novel data about the ligand-Peroxiredoxin interactions that will help considerably the development of potential Peroxiredoxin inhibitors.

Bovine testis hyaluronidase (btHyal) had been shown to have direct effects on cancer cells and to be a useful adjuvant in several medicines. Furthermore this enzyme had been found to be membrane-associated. Thus, in this work, the interactions between btHyal and membranes were analyzed by using lipid monolayers at the air-water interface as a biomimetic membrane system. This allowed us to define the btHyal interactions with two residues of hyaluronic acid (a btHyal substrate), GlcNAc and carboxylic group, which are present in cholesteryl-triethoxy-N-acetylglucosamine (Chol-E3-GlcNAc) and in DPPS, respectively. btHyal bound preferentially Chol-E3-GlcNAc monolayers and showed a decreasing affinity for Chol-E3-GlcNAc-DPPC monolayers containing decreasing amount of glycolipid, suggesting a crucial role of the glycolipid GlcNAc. Furthermore the significant btHyal binding to DPPS was not affected by the presence of free GlcNAc in the subphase. These results and the absence of significant binding of btHyal to pure DPPC monolayer suggest that the protein interacts with the lipid monolayer by mimicking the enzyme-substrate interactions or by electrostatic interactions.

NMR spectroscopy has been used to map the interaction domain on Escherichia coli thioredoxin for the thioredoxin- dependent 5'-adenylylsulfate reductase from Pseudomonas aeruginosa (PaAPR). Seventeen thioredoxin amino acids, all clustered around Cys-32 (the more surface-exposed of the two active-site cysteines), have been located at the PaAPR binding site. The center of the binding domain is dominated by nonpolar amino acids, with a smaller number of charged and polar amino acids located on the periphery of the site. Twelve of the amino acids detected by NMR have non-polar, hydrophobic side chains, including one aromatic amino acid (Trp-31). Four of the thioredoxin amino acids at the PaAPR binding site have polar side chains (Lys-36, Asp-61, Gln-62 and Arg-73), with three of the four having charged side chains. Site-directed mutagenesis experiments have shown that replacement of Lys-36, Asp-61, and Arg-73 and of the absolutely conserved Trp-31 significantly decreases the V(max) for the PaAPR-catalyzed reduction of 5'-adenylylsulfate, with E. coli thioredoxin serving as the electron donor. The most dramatic effect was observed with the W31A variant, which showed no activity as a donor to PaAPR. Although the thiol of the active-site Cys-256 of PaAPR is the point of the initial nucleophilic attack by reduced thioredoxin, mutagenic replacement of Cys-256 by serine has no effect on thioredoxin binding to PaAPR.

Water sonolysis leads to the formation of hydroxyl radicals (OH*). Various techniques are used to detect the OH* production and thus to assess the level of ultrasound-mediated cavitation generated in vitro. In this study, we used terephthalic acid (TA) as an OH* trap. This method is based on the fluorescent properties of hydroxyterephthalic acid (HTA) formed by the reaction of TA with OH* and used as an indicator of the degree of inertial cavitation caused. The experimental system is comprised mainly of a focused piezoelectric ultrasound transmitter and a measurement cell containing 1X PBS/TA diluted solution. In the first part, we aimed to characterize the most appropriate experimental conditions (TA dosimeter solution, irradiation time) in order to optimize the resulting HTA fluorescence values. Then, we could determine that the HTA production increased with the level of the cavitation phenomenon caused by the acoustic power from which OH* production may be estimated.

Experimental NMR diffusion measure on polymers and on globular proteins are presented. These results, complemented with results found in the literature, enable a general description of effective fractal dimension for objects such as small organic molecules, sugars, polymers, DNA, and proteins. Results are compared to computational simulations as well as to theoretical values. A global picture of the diffusion phenomenon emerges from this description. A power law relating molecular mass with diffusion coefficients is described and found to be valid over 4 orders of magnitude. From this law, the fractal dimension of the molecular family can be measured, with experimental values ranging from 1.41 to 2.56 in full agreement with theoretical approaches. Finally, a method for evaluating the molecular mass of unknown solutes is described and implemented as a Web page.

Iturins are a group of antifungal produced by Bacillus subtilis. All are cyclic lipopeptides with seven alpha-amino acids of configuration LDDLLDL and one beta-amino fatty acid. The bacillomycin L is a member of this family and its NMR structure was previously resolved using the sequence Asp-Tyr-Asn-Ser-Gln-Ser-Thr. In this work, we carefully examined the NMR spectra of this compound and detected an error in the sequence. In fact, Asp1 and Gln5 need to be changed into Asn1 and Glu5, which therefore makes it identical to bacillomycin Lc. As a consequence, it now appears that all iturinic peptides with antibiotic activity share the common beta-amino fatty acid 8-L-Asn1-D-Tyr2-D-Asn3 sequence. To better understand the conformational influence of the acidic residue L-Asp1, present, for example in the inactive iturin C, the NMR structure of the synthetic analogue SCP [cyclo (L-Asp1-D-Tyr2-D-Asn3-L-Ser4-L-Gln5-D-Ser6-L-Thr7-beta-Ala8)] was determined and compared with bacillomycin Lc recalculated with the corrected sequence. In both cases, the conformers obtained were separated into two families of similar energy which essentially differ in the number and type of turns. A detailed analysis of both cyclopeptide structures is presented here. In addition, CD and FTIR spectra were performed and confirmed the conformational differences observed by NMR between both cyclopeptides.

Using an in-house fragment NMR library, we identified a set of ligands that bind rabbit muscular creatine kinase, an enzyme involved in key ATP-dependent processes. The ligands docked to the crystal structures of creatine kinase indicated that a phenylfuroic acid could enter into a pocket adjacent to the nucleotide binding site. This fragment served as an anchor to develop in silico a series of potential inhibitors which could partly access the nucleotide binding site. The short synthesis of only four derivatives provided entirely novel hit compounds that reversibly inhibit creatine kinase at micromolar concentrations with a mixed ATP-competitive/noncompetitive mechanism in agreement with the structural model of the inhibited enzyme. These initial biologically active compounds are novel and modular and exploit a new interaction proximate to the ATP binding site.

The NMR structure of the cyclic lipopeptide surfactin from Bacillus subtilis was determined in sodium dodecyl sulfate (SDS) micellar solution. The two negatively charged side chains of surfactin form a polar head opposite to most hydrophobic side chains, accounting for its amphiphilic nature and its strong surfactant properties. Disorder was observed around the fatty acid chain, and 15N relaxation studies were performed to investigate whether it originates from a dynamic phenomenon. A very large exchange contribution to transverse relaxation rate R(2) was effectively observed in this region, indicating slow conformational exchange. Temperature variation and Carr-Purcell-Meiboom-Gill (CPMG) delay variation relaxation studies provided an estimation of the apparent activation energy around 35-43 kJ x mol(-1) and an exchange rate of about 200 ms(-1) for this conformational exchange. 15N relaxation parameters were also recorded in dodecylphosphocholine (DPC) micelles and DMSO. Similar chemical exchange around the fatty acid was found in DPC but not in DMSO, which demonstrates that this phenomenon only occurs in micellar media. Consequently, it may either reflect the disorder observed in our structures determined in SDS or originate from an interaction of the lipopeptide with the detergent, which would be qualitatively similar with an anionic (SDS) or a zwitterionic (DPC) detergent. These structural and dynamics results on surfactin are the first NMR characterization of a lipopeptide incorporated in micelles. Moreover, they provide a model of surfactin determined in a more biomimetic environment than an organic solvent, which could be useful for understanding the molecular mechanism of its biological activity.

Several HLA-DR alleles present the immunodominant HA(306-318) peptide of haemagglutinin of the influenza virus to T cells. NMR data of the peptide in various water solutions exclude any alpha-helix or turn conformations. Circular dichroism and Fourier transform infrared spectroscopies indicate an estimated beta-extended structure in water of 31% and 28%, respectively, with spectra shape similar to the ones observed for beta-sheet containing proteins. The H/D amide exchange suggests a stable length-dependent interchain hydrogen-bonding. The partially beta-extended conformation of HA(306-318) in solution might be close to the one found in HA(306-318)-HLA-DR1 complex. These results suggest different interconverting extended conformations of HA(306-318), depending on the microenvironment of the solution medium. This flexibility emphasizes the ability of some peptides to fit more easily the binding site of several HLA-DR molecules. Similar results were obtained on the HIV P25(263-277) peptide which has been previously shown to be a good DR1 binder. From a vibrational point of view, infrared Amide I frequencies of secondary structures in peptides were ascertained. As previously demonstrated for proteins in solution, Fourier transform infrared and circular dichroism spectroscopies appear to be valuable tools for conformational properties of peptides. Their use may contribute to the detection of peptide conformation-binding relationship which has to be further tested by biochemical and biological studies.

1-Cys peroxiredoxins (1-Cys Prxs) are antioxidant enzymes that catalyze the reduction of hydroperoxides into alcohols using a strictly conserved cysteine. 1-Cys B-Prxs, homologous to human PrxVI, were recently shown to be reactivated by glutathione S-transferase (GST) pi via the formation of a GST-Prx heterodimer and Prx glutathionylation. In contrast, 1-Cys D-Prxs, homologous to human PrxV, are reactivated by the glutaredoxin-glutathione system through an unknown mechanism. To investigate the mechanistic events that mediate the 1-Cys D-Prx regeneration, interaction of the Prx with glutathione was studied by mass spectrometry and NMR. This work reveals that the Prx can be glutathionylated on its active site cysteine. Evidences are reported that the glutathionylation of 1-Cys D-Prx induces the dissociation of the Prx non-covalent homodimer, which can be recovered by reduction with dithiothreitol. This work demonstrates for the first time the existence of a redox-dependent dimer-monomer switch in the Prx family, similar to the decamer-dimer switch for the 2-Cys Prxs.

The interfacial properties of bovine testicular hyaluronidase were investigated by demonstrating the association of hyaluronidase activity with membranes prepared from bovine testis. Protein adsorption to the air/water interface was investigated using surface pressure-area isotherms. In whichever way the interfacial films were obtained (protein injection or deposition), the hyaluronidase exhibited a significant affinity for the air/water interface. The isotherm obtained 180 min after protein injection into a pH 5.3 subphase was similar to the isotherm obtained after spreading the same amount of protein onto the same subphase, indicating that bovine testicular hyaluronidase molecules adopted a similar arrangement and/or conformation at the interface. Increasing the subphase pH from 5.3 to 8 resulted in changes of the protein isotherms. These modifications, which could correspond to the small pH-induced conformational changes observed by Fourier-transform IR spectroscopy, were discussed in relation to the pH influence on the hyaluronidase activity. Adding hyaluronic acid, the enzyme substrate, to the subphase tested the stability of the interfacial properties of hyaluronidase. The presence of hyaluronic acid in the subphase did not modify the protein adsorption and allowed substrate binding to a preformed film of hyaluronidase at pH 5.3, the optimal pH for the enzyme activity. Such effects of hyaluronic acid were not observed when the subphase was constituted of pure water, a medium where the enzyme activity was negligible. These influences of hyaluronic acid were discussed in relation to the modelled structure of bovine testis hyaluronidase where a hydrophobic region was proposed to be opposite of the catalytic site.

Peroxiredoxin systems in plants were demonstrated involved in crucial roles related to reactive oxygenated species (ROS) metabolism and the linked cell signalling to ROS. Peroxiredoxins function as peroxidasic systems that combine at least a reactivating reductant agent like thioredoxins, and sometimes glutaredoxins and glutathion. In the past three years a number of peroxiredoxin structures were solved by crystallography in different experimental crystallisation conditions. The structures have revealed a significant propensity of peroxiredoxins for oligomerism that was confirmed by biophysical studies in solution using NMR and other methods as analytical ultra-centrifugation. These studies showed that quaternary structures of peroxiredoxins involve specific protein-protein interaction interfaces that rely upon the peroxiredoxin types and/or their redox conditions. The protein-protein interactions with the reactivating redoxins essentially lead to transient unstable complexes. We review herein the different protein-protein interactions characterized or deduced from those reports.

Although polyene macrolides are efficient antifungal agents with fungicidal mode of action, their use in medical practice is problematic due to their low solubility and significant human toxicity. In an attempt to address the solubility problem, we have obtained two analogues of nystatin with hydroxy groups at positions C31 and C33 through manipulation of the nystatin polyketide synthase in the producing organism Streptomyces noursei. Structures of the analogues were confirmed by nuclear magnetic resonance (NMR), and their solubility was found to be more than 2000 times higher than that of nystatin. However, both analogues were shown to have lost antifungal activity, implying that the integrity of the hydrophobic polyene region of the nystatin molecule is crucial for the fungicidal action. NMR data and computer modeling performed for the new analogues suggested conformational changes together with a significantly increased structural disorder, which may account for both increased solubility and the loss of activity.

Intracellular accumulation of triphosphorylated derivatives is essential for the cytotoxic activity of nucleoside analogues. Different mechanisms opposing this accumulation have been described. We have investigated the dephosphorylation of monophosphorylated fludarabine (F-ara-AMP) by the purified cytoplasmic 5'-nucleotidase cN-II using HPLC and NMR. These studies clearly showed that cN-II was able to convert F-ara-AMP into its non phosphorylated form, F-ara-A, with a Km in the millimolar range and Vmax = 35 nmol/min/mg, with both methods. Cytoplasmic 5'-nucleotidase cN-II can degrade this clinically useful cytotoxic nucleoside analogue and its overexpression is thus likely to be involved in resistance to this compound.

Thioredoxin f (TRXf) is a key factor in the redox regulation of chloroplastic carbon fixation enzymes, whereas glutathione is an important thiol buffer whose status is modulated by stress conditions. Here, we report specific glutathionylation of TRXf. A conserved cysteine is present in the TRXf primary sequence, in addition to its two active-site cysteines. The additional cysteine becomes glutathionylated when TRXf is exposed to oxidized glutathione or to reduced glutathione plus oxidants. No other chloroplastic TRX, from either Arabidopsis or Chlamydomonas, is glutathionylated under these conditions. Glutathionylation decreases the ability of TRXf to be reduced by ferredoxin-thioredoxin reductase and results in impaired light activation of target enzymes in a reconstituted thylakoid system. Although several mammalian proteins undergoing glutathionylation have already been identified, TRXf is among the first plant proteins found to undergo this posttranslational modification. This report suggests that a crosstalk between the TRX and glutathione systems mediates a previously uncharacterized form of redox signaling in plants in stress conditions.

Glutaredoxins (Grx) represent a large family of glutathione (GSH)-dependent oxidoreductases that catalyse the reduction of disulfides or glutathione mixed disulfide. Grx domains from pathogenic bacteria and plant Grxs have been recently reported to target specific peroxiredoxins (Prxs). The specificity that triggers the interaction between Grx and Prx is poorly understood and is only based on the structure of Haemophilus influenzae Prx-Grx hybrid (hyPrx5). We report here an NMR study of the Populus tremula Grx C4 that targets a P.tremula D-type II Prx. We show that Grx C4 specifically self-associates in a monomer-dimer equilibrium with an apparent K(d) of ca 2.6 mM. Grx C4 homodimer was docked under experimental restraints. The results reveal a novel Grx-Grx interface that is unrelated to the hyPrx5 Grx-Grx dimer interface. Chemical-shift perturbations and 15N spin-relaxation measurements show that the auto-association surface comprises both the active site and the GSH binding site. Reduced GSH is demonstrated to bind reduced Grx with a K(d) of ca 8.6 mM. The potential biological significance of the new Grx-Grx interaction interface is discussed.

In many vertebrate tissues, cytosolic 5'-nucleotidase II (cN-II) either hydrolyses or phosphorylates a number of purine (monophosphorylated) nucleosides through a scheme common to the Haloacid Dehalogenase superfamily members. It possesses a pivotal role in purine cellular metabolism and it acts on anti-tumoural and antiviral nucleoside analogues, thus being of potential therapeutic importance. cN-II is Mg2+-dependent, regulated and stabilised by several factors such as allosteric effectors ATP and 2,3-DPG, although these are not directly involved in the reaction stoichiometry. We review herein the experimental knowledge currently available about this remarkable enzymatic activity.

Peroxiredoxins (Prxs) constitute a family of thiol peroxidases that reduce hydrogen peroxide, peroxinitrite, and hydroperoxides using a strictly conserved cysteine. Very abundant in all organisms, Prxs are produced as diverse isoforms characterized by different catalytic mechanisms and various thiol-containing reducing agents. The oligomeric state of Prxs and the link with their functionality is a subject of intensive research. We present here a combined X-ray and nuclear magnetic resonance (NMR) study of a plant Prx that belongs to the D-Prx (type II) subfamily. The Populus trichocarpa Prx is the first Prx shown to be regenerated in vitro by both the glutaredoxin and thioredoxin systems. The crystal structure and solution NMR provide evidence that the reduced protein is a specific noncovalent homodimer both in the crystal and in solution. The dimer interface is roughly perpendicular to the plane of the central beta sheet and differs from the interface of A- and B-Prx dimers, where proteins associate in the plane parallel to the beta sheet. The homodimer interface involves residues strongly conserved in the D (type II) Prxs, suggesting that all Prxs of this family can homodimerize. The study provides a new insight into the Prx oligomerism and the basis for protein-protein and enzyme-substrate interaction studies by NMR.

The gram-positive bacterium Streptomyces noursei ATCC 11455 produces a complex mixture of polyene macrolides generally termed nystatins. Although the structures for nystatins A(1) and A(3) have been reported, the identities of other components of the nystatin complex remain obscure. Analyses of the culture extract from the S. noursei wild type revealed the presence of several nystatin-related compounds for which chemical structures could be suggested on the basis of their molecular weights, their UV spectra, and knowledge of the nystatin biosynthetic pathway. Nuclear magnetic resonance (NMR) studies with one of these polyene macrolides identified it as a nystatin analogue containing a mycarose moiety at C-35. A similar investigation was performed with the culture extract of the ERD44 mutant, which has a genetically altered polyketide synthase (PKS) NysC and which was previously shown to produce a heptaene nystatin analogue. The latter compound, tentatively named S44HP, and its derivative, which contains two deoxysugar moieties, were purified; and their structures were confirmed by NMR analysis. Nystatin analogues with an expanded macrolactone ring were also observed in the extract of the ERD44 mutant, suggesting that the altered PKS can "stutter" during the polyketide chain assembly. These data provide new insights into the biosynthesis of polyene macrolide antibiotics and the functionalities of PKSs and post-PKS modification enzymes.

Delta-conotoxin EVIA, from Conus ermineus, is a 32-residue polypeptide cross-linked by three disulfide bonds forming a four-loop framework. delta-Conotoxin EVIA is the first conotoxin known to inhibit sodium channel inactivation in neuronal membranes from amphibians and mammals (subtypes rNa(v)1.2a, rNa(v)1.3, and rNa(v)1.6), without affecting rat skeletal muscle (subtype rNa(v)1.4) and human cardiac muscle (subtype hNa(v)1.5) sodium channel (Barbier, J., Lamthanh, H., Le Gall, F., Favreau, P., Benoit, E., Chen, H., Gilles, N., Ilan, N., Heinemann, S. F., Gordon, D., Ménez, A., and Molgó, J. (2004) J. Biol. Chem. 279, 4680-4685). Its structure was solved by NMR and is characterized by a 1:1 cis/trans isomerism of the Leu(12)-Pro(13) peptide bond in slow exchange on the NMR time scale. The structure of both cis and trans isomers could be calculated separately. The isomerism occurs within a specific long disordered loop 2, including residues 11-19. These contribute to an important hydrophobic patch on the surface of the toxin. The rest of the structure matches the "inhibitor cystine-knot motif" of conotoxins from the "O superfamily" with a high structural order. To probe a possible functional role of the Leu(12)-Pro(13) cis/trans isomerism, a Pro(13) --> Ala delta-conotoxin EVIA was synthesized and shown to exist only as a trans isomer. P13A delta-conotoxin EVIA was estimated only two times less active than the wild-type EVIA in binding competition to rat brain synaptosomes and when injected intracerebroventricularly into mice.

NMR spectroscopy has evolved dramatically over the past 15 years, establishing a new, reliable methodology for studying biomacromolecules at atomic resolution. The three-dimensional structure and dynamics of a biomolecule or a biomolecular complex is only one of the main types of information available using NMR. The spectral assignment to the specific nuclei of a biostructure is a very precise reflection of their electronic environment. Any change in this environment due to a structural change, the binding of a ligand or the redox state of a redox cofactor, will be very sensitively reported by changes in the different NMR parameters. The capabilities of the NMR method are currently expanding dramatically and it is turning into a powerful means to study biosystems dynamically in exchange between different conformations, exchanging ligands, transient complexes, or the activation/inhibition of regulated enzymes. We review here several NMR studies that have appeared during the past 5 or 6 years in the field of redox proteins of plants, yeasts and photosynthetic bacteria. These new results illustrate the recent biomolecular NMR evolution and provide new physiological models for understanding the different types of electron transfer, including glutaredoxins, thioredoxins and their dependent enzymes, the ferredoxin-NADP oxidoreductase complex, flavodoxins, the plastocyanin-cytochrome f complex, and cytochromes c.

Peroxiredoxins (Prx's) are a superfamily of thiol-specific antioxidant proteins present in all organisms and involved in the hydroperoxide detoxification of the cell. The catalytic cysteine of Prx's reduces hydroperoxides and is transformed into a transient sulfenic acid (Cys-SOH). At high hydroperoxide concentration, the sulfenic acid can be overoxidized into a sulfinate, or even a sulfonate. We present here the first peroxiredoxin characterization by solution NMR of the Saccharomyces cerevisiae alkylhydroperoxide reductase (Ahp1) in its reduced and in vitro overoxidized forms. NMR (15)N relaxation data and ultracentrifugation experiments indicate that the protein behaves principally as a homodimer (2 x 19 kDa) in solution, regardless of the redox state. In vitro treatment of Ahp1 by a large excess of tBuOOH leads to an inactive form, with the catalytic cysteine overoxidized into sulfonate, as demonstrated by (13)C NMR. Depending on the amino acid sequence of their active site, Prx's are classified into five different families. In this classification, Ahp1 is a member of the scarcely studied D-type Prx's. Ahp1 is unique among the D-type Prx's in its ability to form an intermolecular disulfide. The peptidic sequence of Ahp1 was analyzed and compared to other D-type Prx sequences.

The purpose of this work was to screen clinical isolates of actinomycetes producing nonpolyenic antifungals. This choice was made to limit the problem of rediscovery of well-known antifungal families, especially polyenic antifungals. One hundred and ten strains were tested, using two diffusion methods and two test media, against three yeast species and three filamentous fungi. Among 54 strains (49%) showing antifungal activity, five strains belonging to the genus Streptomyces were active against all test organisms and appeared promising. These results indicate that clinical and environmental isolates of actinomycetes could be an interesting source of antifungal bioactive substances. The production of nonpolyenic antifungal substances by these five active isolates was investigated using several criteria: antibacterial activity, ergosterol inhibition, and UV-visible spectra of active extracts. One active strain responded to all three selection criteria and produced potentially nonpolyenic antifungal metabolites. This strain was retained for further investigation, in particular, purification, structure elucidation, and mechanism of action of the active product.

Reaction-induced infrared difference spectroscopy (RIDS) has been used to investigate the nature of interactions of human annexin A6 (ANXA6) with nucleotides. RIDS results for ANXA6, obtained after the photorelease of GTP-gamma-S, ATP, or P(i) from the respective caged compounds, were identical, suggesting that the interactions between the nucleotide and ANXA6 were dominated by the phosphate groups. Phosphate-induced structural changes in ANXA6 were small and affected only seven or eight amino acid residues. The GTP fluorescent analogue, 2'(3')-O-(2,4,6-trinitrophenyl)guanosine 5'-triphosphate (TNP-GTP), quenched tryptophan fluorescence of ANXA6 when bound to the protein. A binding stoichiometry of 1 mol of nucleotide/mol ANXA6 was established with a K(D) value of 2.8 microM for TNP-GTP. The bands observed on RIDS of ANXA6 halves (e.g., N-terminal half, ANXA6a, and C-terminal half, ANXA6b) were similar to those of the whole molecule. However, their amplitudes were smaller by a factor of 2 compared to those of whole ANXA6. TNP-GTP bound to both fragments of ANXA6 with a stoichiometry of 0.5 mol/mol. However, the binding affinities of ANXA6a and ANXA6b differed from that of ANXA6. Simulated molecular modeling revealed a nucleotide-binding site which was distributed in two distinct domains. Residues K296, Y297, K598, and K644 of ANXA6 were less than 3 A from the bound phosphate groups of either GTP or ATP. The presence of two identical sequences in ANXA6 with the F-X-X-K-Y-D/E-K-S-L motif, located in the middle of ANXA6, at residues 293-301 (within ANXA6a) and at 641-649 (within ANXA6b), suggested that the F-X-X-K-Y-D/E-K-S-L motif was the putative sequence in ANXA6 for nucleotide binding.

The sequencing of the genome of Arabidopsis thaliana revealed that this plant contained numerous isoforms of thioredoxin (Trx), a protein involved in thiol-disulfide exchanges. On the basis of sequence comparison, seven putative chloroplastic Trxs have been identified, four belonging to the m-type, two belonging to the f-type, and one belonging to a new x-type. In the present work, these isoforms were produced and purified as recombinant proteins without their putative transit peptides. Their activities were tested with two known chloroplast thioredoxin targets: NADP-malate dehydrogenase and fructose-1,6-bisphosphatase and also with a chloroplastic 2-Cys peroxiredoxin. The study confirms the strict specificity of fructose-bisphosphatase for Trx f, reveals that some Trxs are unable to activate NADP-malate dehydrogenase, and shows that the new x-type is the most efficient substrate for peroxiredoxin while being inactive toward the two other targets. This suggests that this isoform might be specifically involved in resistance against oxidative stress. Three-dimensional modeling shows that one of the m-type Trxs, Trx m3, which has no activity with any of the three targets, exhibits a negatively charged surface surrounding the active site. A green fluorescent protein approach confirms the plastidial localization of these Trxs.

Glycosylated polyene macrolide antibiotics, as nystatins and amphotericins, are amphiphilic structures known to exert antifungal activity by disrupting the fungal cell membrane, leading to leakage of cellular materials, and cell death. This membrane disruption is strongly influenced by the presence and the exact nature of the membrane sterols. The solution structures of five representative glycosylated members, three tetraenes (pimaricin, nystatin A1 and rimocidin) and two heptaenes (candidin and vacidin A) have been calculated using geometric restraints derived from 1H-NMR data and random searches of their conformational space. Despite a different apparent structural order, the NMR solutions structure indicate that the hydroxyl groups all clustered on one side of the rod-shaped structures, and the glycosyl moieties are structurally conserved both in their conformation and their apparent order. The molecular structures afford an understanding of their selective interaction with the membrane sterols and the design of new polyene macrolides with improved activities.

Two novel toxins, Lqh6 and Lqh7, isolated from the venom of the scorpion Leiurus quinquestriatus hebraeus, have in their sequence a molecular signature (8Q/KPE10) associated with a recently defined group of alpha-toxins that target Na channels, namely the alpha-like toxins [reviewed in Gordon, D., Savarin, P., Gurevitz, M. & Zinn-Justin, S. (1998) J. Toxicol. Toxin Rev. 17, 131-159]. Lqh6 and Lqh7 are highly toxic to insects and mice, and inhibit the binding of alpha-toxins to cockroach neuronal membranes. Although they kill rodents by intracerebroventricular injection, they do not inhibit the binding of antimammal alpha-toxins (e.g. Lqh2) to rat brain synaptosomes, not even at high concentrations. Furthermore, in voltage-clamp experiments, rat brain Na channels IIA (rNav1.2A) expressed in Xenopus oocytes are not affected by Lqh6 nor by Lqh7 below 3 micro m. In contrast, muscular Na channels (rNav1.4 and hNav1.5) expressed in the same cells respond to nanomolar concentrations of Lqh6 and Lqh7 by slowing of Na current inactivation and a leftward shift of the peak conductance-voltage curve. The structural and pharmacological properties of the new toxins are compared to those of other scorpion alpha-toxins in order to re-examine the hallmarks previously set for the alpha-like toxin group.

Chloroplast NADP-dependent malate dehydrogenase (NADP-MDH, EC 1.1.1.82) is inactive in the dark and activated in the light via a reduction of specific disulfides by thiol-disulfide interchange with thioredoxin, reduced by the photosynthetic electron transfer. Compared to the constitutively active NAD-dependent forms, NADP-MDH exhibits two regulatory disulfides per subunit, one located in an N-terminal extension and the other in a C-terminal extension. Convergent information gathered from biochemical, site-directed mutagenesis and structural approaches allowed to solve almost completely the activation mechanism. In the oxidized enzyme, the C-terminal extension is pulled back by the disulfide bridge toward the active-site cleft where the penultimate C-terminal glutamate interacts with one of the arginines involved in substrate binding, thus acting as an internal inhibitor obstructing the access of oxaloacetate. The N-terminal extensions are located at the subunit interface area and rigidify the overall structure of the dimer. Their reduction by reduced thioredoxin triggers a conformational change of the active site towards high-activity conformation, whereas the reduction of the C-terminal bridge expells the C-terminal end from the active site, thus opening the way for the substrate.

Plipastatins A and B are antifungal antibiotics belonging to a family of lipopeptides capable of inhibiting phospholipase A(2) (PLA(2)) and are biosynthesised under certain circumstances by Bacillus subtilis. U-(15)N plipastatins A and B were obtained from cultures of the strain NCIB 8872 on a Landy medium modified for stable-isotope labelling by the substitution of the L-glutamic acid used as the sole nitrogen source, by (15)NH(4)Cl. These two lipo-decapeptides, lactonised by esterification of the Ile10 C-terminus with the phenolic hydroxyl of Tyr3, differ only by a D-Ala (plipastatin A)/D-Val (plipastatin B) substitution at the position 6. The (1)H- and (15)N-nuclear magnetic resonance (NMR) signals of a 4:6 mixture of plipastatins A and B were unambiguously assigned and their structures in dimethylsulfoxide solution were calculated on the basis of a set of NMR-derived restraints. Plipastatins A and B are well-defined structures in solution stabilised by a type 1 beta-turn comprising residues 6-9 and several other specific hydrogen bonds. The structures afford a first molecular basis for the future studies of their biological activities both in lipidic layers or on PLA(2).

Chloroplast thioredoxin m from the green alga Chlamydomomas reinhardtii is very efficiently reduced in vitro and in vivo in the presence of photoreduced ferredoxin and a ferredoxin dependent ferredoxin-thioredoxin reductase. Once reduced, thioredoxin m has the capability to quickly activate the NADP malate dehydrogenase (EC 1.1.1.82) a regulatory enzyme involved in an energy-dependent assimilation of carbon dioxide in C4 plants. This activation is the result of the reduction of two disulfide bridges by thioredoxin m, that are located at the N- and C-terminii of the NADP malate dehydrogenase. The molecular structure of thioredoxin m was solved using NMR and compared to other known thioredoxins. Thioredoxin m belongs to the prokaryotic type of thioredoxin, which is divergent from the eukaryotic-type thioredoxins also represented in plants by the h (cytosolic) and f (chloroplastic) types of thioredoxins. The dynamics of the molecule have been assessed using (15)N relaxation data and are found to correlate well with regions of disorder found in the calculated NMR ensemble. The results obtained provide a novel basis to interpret the thioredoxin dependence of the activation of chloroplast NADP-malate dehydrogenase. The specific catalytic mechanism that takes place in the active site of thioredoxins is also discussed on the basis of the recent new understanding and especially in the light of the dual general acid-base catalysis exerted on the two cysteines of the redox active site. It is proposed that the two cysteines of the redox active site may insulate each other from solvent attack by specific packing of invariable hydrophobic amino acids.

The disruption of the two thioredoxin genes in Saccharomyces cerevisiae leads to a complex phenotype, including the inability to use methionine sulfoxide as sulfur source, modified cell cycle parameters, reduced H(2)O(2) tolerance, and inability to use sulfate as sulfur source. Expression of one of the multiple Arabidopsis thaliana thioredoxins h in this mutant complements only some aspects of the phenotype, depending on the expressed thioredoxin: AtTRX2 or AtTRX3 induce methionine sulfoxide assimilation and restore a normal cell cycle. In addition AtTRX2 also confers growth on sulfate but no H(2)O(2) tolerance. In contrast, AtTRX3 does not confer growth on sulfate but induces H(2)O(2) tolerance. We have constructed hybrid proteins between these two thioredoxins and show that all information necessary for sulfate assimilation is present in the C-terminal part of AtTRX2, whereas some information needed for H(2)O(2) tolerance is located in the N-terminal part of AtTRX3. In addition, mutation of the atypical redox active site WCPPC to the classical site WCGPC restores some growth on sulfate. All these data suggest that the multiple Arabidopsis thioredoxins h originate from a totipotent ancestor with all the determinants necessary for interaction with the different thioredoxin target proteins. After duplications each member evolved by losing or masking some of the determinants.

The alpha-like toxin from the venom of the scorpion Leiurus quinquestriatus hebraeus (Lqh III) binds with high affinity to receptor site 3 on insect sodium channels but does not bind to rat brain synaptosomes. The binding affinity of Lqh III to cockroach neuronal membranes was fivefold higher at pH 6.5 than at pH 7.5. This correlated with an increase in the electropositive charge on the toxin surface resulting from protonation of its four histidines. Radioiodination of Tyr(14) of Lqh III abolished its binding to locust but not cockroach sodium channels, whereas the noniodinated toxin bound equally well to both neuronal preparations. Radioiodination of Tyr(10) or Tyr(21) of the structurally similar alpha-toxin from L. quinquestriatus hebraeus (LqhalphaIT), as well as their substitution by phenylalanine, had only minor effects on binding to cockroach neuronal membranes. However, substitution of Tyr(21), but not Tyr(14), by leucine decreased the binding affinity of LqhalphaIT approximately 87-fold. Thus, Tyr(14) is involved in the bioactivity of Lqh III to locust receptor site 3 and is not crucial for the binding of LqhalphaIT to this site. In turn, the aromatic ring of Tyr(21) takes part in the bioactivity of LqhalphaIT to insects. These results highlight subtle architectural variations between locust and cockroach receptor site 3, in addition to previous results demonstrating the competence of Lqh III to differentiate between insect and mammalian sodium channel subtypes.

Thioredoxins are small proteins found in all living organisms. We have previously reported that Chlamydomonas reinhardtii thioredoxin h exhibited differences both in its absorption spectrum and its aggregation properties compared to thioredoxin m. In this paper, we demonstrate, by site-directed mutagenesis, that the particularity of the absorption spectrum is linked to the presence of an additional tryptophan residue in the h isoform. The pH and temperature dependence of the aggregation of both thioredoxins has been investigated. Our results indicate that the aggregation of TRX is highly dependent on pH and that the differences between the two TRX isoforms are linked to distinct pH dependencies. We have also analyzed the pH and temperature dependence of 12 distinct variants of TRX engineered by site-directed mutagenesis. The results obtained indicate that the differences in the hydrophobic core of the two TRX isoforms do not account for the differences of aggregation. On the other hand, we show the importance of His-109 as well as the second active site cysteine, Cys-39 in the aggregation mechanism.

The chloroplastic NADP-dependent malate dehydrogenase (NADP-MDH) catalyzing the reduction of oxaloacetate into L-malate is regulated by light. Its activation results from the thioredoxin-mediated reduction of two disulfides, located, respectively, in N- and C-terminal sequence extensions typical of all NADP-dependent light-regulated forms. Site-directed mutagenesis studies and the resolution of the three-dimensional structure of the oxidized (inactive) Sorghum vulgare enzyme showed that the C-terminal Cys(365)-Cys(377) disulfide constrains the C-terminal extension to fold into the active site where it acts as an internal inhibitor. In the present study, two-dimensional proton NMR spectra of an engineered NADP-MDH rendered monomeric by a 33-amino acid deletion at the N terminus (38 kDa) revealed that a 15-amino acid-long C-terminal peptide (Ala(375) to C-terminal Val(389)) acquired an increased mobility upon reduction, allowing its direct sequence-specific NMR assignment. The location of the flexible peptide in the sequence suggests that the first part of the C-terminal peptide is still folded near the core of the enzyme, so that cysteines 365 and 377 remain in proximity to allow for an efficient reoxidation/inactivation of the enzyme.

A synthetic peptide MQVTMKSSAVSGQRVGGARVATRSVRRAQLQV corresponding to the 32 amino acid chloroplast transit sequence of the ribulose bisphosphatase carboxylase/oxygenase activase preprotein from Chlamydomonas reinhardtii, required for translocation through the envelope of the chloroplast, has been characterized structurally using CD and NMR under the same experimental conditions as used previously for the 32 amino acid presequence of preferredoxin from the same organism [Lancelin, J.-M., Bally, I., Arlaud, G. J., Blackledge, M., Gans, P., Stein, M. & Jacquot, J.-P. (1994) FEBS Lett. 343, 261-266]. The peptide is found to undergo a conformational transition in aqueous 2,2,2-trifluoroethanol, characterized by three turns of amphiphilic alpha-helix in the C-terminal region preceded by a disordered coil in the N-terminal region. Compared with the preferredoxin transit peptide, the helical and coiled domains are arranged in the reverse order along the peptide sequence, but the positively charged groups are distributed analogously as well as the hydrophobic residues within the amphiphilic alpha-helix. It is proposed that such coil-helix or helix-coil motifs, occasionally repeated, could be an intrinsic structural feature of chloroplastic transit peptides, adapted to the proper translocase and possibly to each nuclear-encoded chloroplast preproteins. This feature may distinguish chloroplastic transit sequences from the other organelle-targeting peptides in the eukaryotic green alga C. reinhardtii, particularly the mitochondrial transit sequences.

The antifungal antibiotic lipopeptide bacillomycin L [cyclo-(L-Asp1-D-Tyr2-D-Asn3-L-Ser4-L-Gln5-D-Ser6++ +-L-Thr7-beta-amino fatty acid)] from Bacillus subtilis belongs to the iturinic family of antifungal agents and acts with a strict sterol-phospholipid dependence on biomembranes. This antibiotic has been analysed using solution NMR spectroscopy in its native active form and its inactive (L-Asp1, D-Tyr2) di-O-methylated form. The structures were calculated under NMR-derived restraints using molecular-dynamic simulated-annealing protocols starting from a random array of atoms. The structure of the native antibiotic is spread over different conformers in which two families are recognized. It was found that most structures have dihedral phi and psi angles defining a type-II' beta-turn including amino acids 5-8, in certain cases stabilized by a 8HN-5CO hydrogen bond, whereas a minority of structures adopt an inverse gamma-turn including amino acids 6-8, stabilized in all cases by an 8HN-6CO hydrogen bond. The di-O-methylation of L-Asp1 and D-Tyr2, an amino acid strictly conserved within the iturinic group of antibiotics, does not induce major differences in the NMR spectra and in the NMR structures. The results are discussed in relation to the specific loss of interaction with sterols when the native antifungal bacillomycin L is methylated on the conserved D-Tyr2 position.