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BackgroundTransposons are mobile genetic elements that colonize genomes and drive their plasticity in all organisms. DNA transposon-encoded transposases bind to the ends of their cognate transposons and catalyze their movement. In some cases, exaptation of transposon genes has allowed novel cellular functions to emerge. The PiggyMac (Pgm) endonuclease of the ciliateParamecium tetraureliais a domesticated transposase from the PiggyBac family. It carries a core catalytic domain typical of PiggyBac-related transposases and a short cysteine-rich domain (CRD), flanked by N- and C-terminal extensions. During sexual processes Pgm catalyzes programmed genome rearrangements (PGR) that eliminate ~ 30% of germline DNA from the somatic genome at each generation. How Pgm recognizes its DNA cleavage sites in chromatin is unclear and the structure-function relationships of its different domains have remained elusive.ResultsWe provide insight into Pgm structure by determining the fold adopted by its CRD, an essential domain required for PGR. Using Nuclear Magnetic Resonance, we show that the Pgm CRD binds two Zn2+ions and forms an unusual binuclear cross-brace zinc finger, with a circularly permutated treble-clef fold flanked by two flexible arms. The Pgm CRD structure clearly differs from that of several other PiggyBac-related transposases, among which is the well-studied PB transposase fromTrichoplusia ni. Instead, the arrangement of cysteines and histidines in the primary sequence of the Pgm CRD resembles that of active transposases frompiggyBac-like elements found in other species and of human PiggyBac-derived domesticated transposases. We show that, unlike the PB CRD, the Pgm CRD does not bind DNA. Instead, it interacts weakly with the N-terminus of histone H3, whatever its lysine methylation state.ConclusionsThe present study points to the structural diversity of the CRD among transposases from the PiggyBac family and their domesticated derivatives, and highlights the diverse interactions this domain may establish with chromatin, from sequence-specific DNA binding to contacts with histone tails. Our data suggest that the Pgm CRD fold, whose unusual arrangement of cysteines and histidines is found in all PiggyBac-related domesticated transposases fromParameciumandTetrahymena, was already present in the ancestral active transposase that gave rise to ciliate domesticated proteins.

International audience; The piggyBac transposase (PB) is distinguished by its activity and utility in genome engineering, especially in humans where it has highly promising therapeutic potential. Little is known, however, about the structure-function relationships of the different domains of PB. Here, we demonstrate in vitro and in vivo that its C-terminal Cysteine-Rich Domain (CRD) is essential for DNA breakage, joining and transposition and that it binds to specific DNA sequences in the left and right transposon ends, and to an additional unexpectedly internal site at the left end. Using NMR, we show that the CRD adopts the specific fold of the cross-brace zinc finger protein family. We determine the interaction interfaces between the CRD and its target, the 5'-TGCGT-3'/3'-ACGCA-5' motifs found in the left, left internal and right transposon ends, and use NMR results to propose docking models for the complex, which are consistent with our site-directed mutagenesis data. Our results provide support for a model of the PB/DNA interactions in the context of the transpososome, which will be useful for the rational design of PB mutants with increased activity.

International audience; Proteins of the Bcl-2 family are key targets in anticancer drug discovery. Disrupting the interaction between anti and pro-apoptotic members of this protein family was the approach chosen in this study to restore apoptosis. Thus, a biological screening on the modulation of the Bcl-xL/Bak and Mcl-1/Bid interactions permitted the selection of Knema hookeriana for further phytochemical investigations. The ethyl acetate extract from the stem bark led to the isolation of six new compounds, three acetophenone derivatives (1-3) and three anacardic acid derivatives (4-6), along with four known anacardic acids (7-10) and two cardanols (11, 12). Their structures were elucidated by 1D and 2D NMR analysis in combination with HRMS experiments. The ability of these compounds to antagonize Bcl-xL/Bak and Mcl-1/Bid association was determined, using a protein protein interaction assay, but only anacardic acid derivatives (4-10) exhibited significant binding properties, with K-i values ranging from 0.2 to 18 mu M. Protein ligand NMR experiments further revealed that anacardic acid 9, the most active compound, does not interact with the anti-apoptotic proteins Bcl-xL and Mcl-1 but instead interacts with pro-apoptotic protein Bid.

International audience; Diflavin reductases are bidomain electron transfer proteins in which structural reorientation is necessary to account for the various intramolecular and intermolecular electron transfer steps. Using small-angle x-ray scattering and nuclear magnetic resonance data, we describe the conformational free-energy landscape of the NADPH-cytochrome P450 reductase (CPR), a typical bidomain redox enzyme composed of two covalently-bound flavin domains, under various experimental conditions. The CPR enzyme exists in a salt- and pH-dependent rapid equilibrium between a previously described rigid, locked state and a newly characterized, highly flexible, unlocked state. We further establish that maximal electron flux through CPR is conditioned by adjustable stability of the locked-state domain interface under resting conditions. This is rationalized by a kinetic scheme coupling rapid conformational sampling and slow chemical reaction rates. Regulated domain interface stability associated with fast stochastic domain contacts during the catalytic cycle thus provides, to our knowledge, a new paradigm for improving our understanding of multidomain enzyme function.

Microcin J25 (MccJ25) has emerged as an excellent model to understand the maturation of ribosomal precursor peptides into the entangled lasso fold. MccJ25 biosynthesis relies on the post-translational modification of the precursor McjA by the ATP-dependent protease McjB and the lactam synthetase McjC. Here, using NMR spectroscopy, we showed that McjA is an intrinsically disordered protein without detectable conformational preference, which emphasizes the active role of the maturation machinery on the three-dimensional folding of MccJ25. We further showed that the N-terminal region of the leader peptide is involved in interaction with both maturation enzymes and identified a predominant interaction of V43-S55 in the core McjA sequence with McjC. Moreover, we demonstrated that residues K23-Q34 in the N-terminal McjA leader peptide tend to adopt a helical conformation in the presence of membrane mimics, implying a role in directing McjA to the membrane in the vicinity of the lasso synthetase/export machinery. These data provide valuable insights into the initial molecular recognition steps in the MccJ25 maturation process.

International audience; Mapping the conformational landscape of G protein-coupled receptors (GPCRs), and in particular how this landscape is modulated by the membrane environment, is required to gain a clear picture of how signaling proceeds. To this end, we have developed an original strategy based on solution-state nuclear magnetic resonance combined with an efficient isotope labeling scheme. This strategy was applied to a typical GPCR, the leukotriene B4 receptor BLT2, reconstituted in a lipid bilayer. Because of this, we are able to provide direct evidence that BLT2 explores a complex landscape that includes four different conformational states for the unliganded receptor. The relative distribution of the different states is modulated by ligands and the sterol content of the membrane, in parallel with the changes in the ability of the receptor to activate its cognate G protein. This demonstrates a conformational coupling between the agonist and the membrane environment that is likely to be fundamental for GPCR signaling.

The NADPH cytochrome P450 reductase (CPR), a diflavin enzyme, catalyzes the electron transfer (ET) from NADPH to the substrate P450. The crystal structures of mammalian and yeast CPRs show a compact organization for the two domains containing FMN (flavin mononucleotide) and FAD (flavin adenine dinucleotide), with a short interflavin distance consistent with fast ET from the NADPH-reduced FAD to the second flavin FMN. This conformation, referred as closed, contrasts with the alternative opened or extended domain arrangements recently described for partially reduced or mutant CPR. Internal domain flexibility in this enzyme is indeed necessary to account for the apparently conflicting requirements of having FMN flavin accessible to both the FAD and the substrate P450 at the same interface. However, how interdomain dynamics influence internal and external ETs in CPR is still largely unknown. Here, we used NMR techniques to explore the global, domain-specific and residue-specific structural and dynamic properties of the nucleotide-free human CPR in solution in its oxidized state. Based on the backbone resonance assignment of this 70-kDa protein, we collected residue-specific 15N relaxation and 1H- 15N residual dipolar couplings. Surprisingly and in contrast with previous studies, the analysis of these NMR data revealed that the CPR exists in a unique and predominant conformation that highly resembles the closed conformation observed in the crystalline state. Based on our findings and the previous observations of conformational equilibria of the CPR in partially reduced states, we propose that the large-scale conformational transitions of the CPR during the catalytic cycle are tightly controlled to ensure optimal electron delivery. © 2012 Published by Elsevier Ltd.

International audience; Human mitoNEET (mNT) is the first identified Fe-S protein of the mammalian outer mitochondrial membrane. Recently, mNT has been implicated in cytosolic Fe-S repair of a key regulator of cellular iron homeostasis. Here, we aimed to decipher the mechanism by which mNT triggers its Fe-S repair capacity. By using tightly controlled reactions combined with complementary spectroscopic approaches, we have determined the differential roles played by both the redox state of the mNT cluster and dioxygen in cluster transfer and protein stability. We unambiguously demonstrated that only the oxidized state of the mNT cluster triggers cluster transfer to a generic acceptor protein and that dioxygen is neither required for the cluster transfer reaction nor does it affect the transfer rate. In the absence of apo-acceptors, a large fraction of the oxidized holo-mNT form is converted back to reduced holo-mNT under low oxygen tension. Reduced holo-mNT, which holds a [2Fe-2S](+)with a global protein fold similar to that of the oxidized form is, by contrast, resistant in losing its cluster or in transferring it. Our findings thus demonstrate that mNT uses an iron-based redox switch mechanism to regulate the transfer of its cluster. The oxidized state is the "active state," which reacts promptly to initiate Fe-S transfer independently of dioxygen, whereas the reduced state is a "dormant form." Finally, we propose that the redox-sensing function of mNT is a key component of the cellular adaptive response to help stress-sensitive Fe-S proteins recover from oxidative injury.

International audience; CGC-19, a 14 kDa proteic constituent of a non ribosomal peptide synthetase implicated in the biosynthesis of a secondary metabolite in Streptomyces ambofaciens, has been isotopically enriched and recombinantly expressed. Its nearly complete (1)H, (13)C and (15)N resonance assignment is reported hereunder.

International audience; Microcin J25 is a potent antibacterial peptide produced by Escherichia coli AY25. It displays a lasso structure, which consists of a knot involving an N-terminal macrolactam ring through which the C-terminal tail is threaded and sterically trapped. In this study, we rationally designed and performed site-specific mutations in order to pinpoint the sequence determinants of the lasso topology. Structures of the resulting variants were analysed by a combination of methods (mass spectrometry, NMR spectroscopy, enzymatic digestion), and correlated to the antibacterial activity. The selected mutations resulted in the production of branched-cyclic or lasso variants. The C-terminal residues below the ring (Tyr20, Gly21) and the size of the macrolactam ring were revealed to be critical for both the lasso scaffold and bioactivity, while shortening the loop region (Tyr9-Ser18) or extending the C-terminal tail below the ring did not alter the lasso structure, but differentially affected the antibacterial activity. These results provide new insights for the bioengineering of antibacterial agents using a lasso peptide as template.

β-Thymosin (βT) and WH2 domains are widespread, intrinsically disordered actin-binding peptides that display significant sequence variability and different regulations of actin self-assembly in motile and morphogenetic processes. Here, we reveal the structural mechanisms by which, in their 1:1 stoichiometric complexes with actin, they either inhibit assembly by sequestering actin monomers like Thymosin-β4, or enhance motility by directing polarized filament assembly like Ciboulot βT. We combined mutational, functional or structural analysis by X-ray crystallography, SAXS (small angle X-ray scattering) and NMR on Thymosin-β4, Ciboulot, TetraThymosinβ and the long WH2 domain of WASP-interacting protein. The latter sequesters G-actin with the same molecular mechanisms as Thymosin-β4. Functionally different βT/WH2 domains differ by distinct dynamics of their C-terminal half interactions with G-actin pointed face. These C-terminal interaction dynamics are controlled by the strength of electrostatic interactions with G-actin. At physiological ionic strength, a single salt bridge with actin located next to their central LKKT/V motif induces G-actin sequestration in both isolated long βT and WH2 domains. The results open perspectives for elucidating the functions of βT/WH2 domains in other modular proteins.

The beta-thymosin/WH2 actin-binding module shows an amazing adaptation to multifunctionality. The beta-thymosins are genuine G-actin sequesterers of moderate affinity for G-actin, allowing an efficient regulation of the G-actin/F-actin ratio in cells by amplifying changes in the critical concentration for filament assembly. In contrast, the first beta-thymosin domain of the protein Ciboulot makes with G-actin a complex that supports filament growth, such as profilin-actin. We illustrate how the use of engineered chimeric proteins, actin-binding and polymerization assays, crystallographic, NMR, and SAXS structural approaches complement each other to decipher the molecular basis for the functional versatility of these intrinsically disordered domains when they form various 1:1 complexes with G-actin. Multifunctionality is expanded in tandem repeats of WH2 domains present in WASP family proteins and proteins involved in axis patterning like Cordon-Bleu and Spire. The tandem repeats generate new functions such as filament nucleation and severing, as well as barbed end binding, which add up to the G-actin sequestering activity. Novel regulation pathways in actin assembly emerge from these additional activities.

An NMR method is described which should provide a rapid means for determining and assigning antiparallel sheets and helices in small proteins. It begins by locating apparent NOESY crosspeaks which suggest the presence of the secondary structure; this is followed by searches for MCD patterns (Englander & Wand (1987) Biochemistry 22, 5953) which are characteristic of these structures. As a result, only spin-systems of the amino acids within the secondary structure need to be defined. A triple-stranded, antiparallel sheet and a helix have been found and assigned for both alpha-cobratoxin and the scorpion toxin AaH III.

International audience; Lasso peptides are bacterial ribosomally synthesized and post-translationally modified peptides. They have sparked increasing interest in peptide-based drug development because of their compact, interlocked structure, which offers superior stability and protein-binding capacity. Disulfide bond-containing lasso peptides are rare and exhibit highly sought-after activities. In an effort to expand the repertoire of such molecules, we heterologously expressed, in Streptomyces coelicolor, the gene cluster encoding sviceucin, a type I lasso peptide with two disulfide bridges originating from Streptomyces sviceus, which allowed it to be fully characterized. Sviceucin and its reduced forms were characterized by mass spectrometry and peptidase digestion. The three-dimensional structure of sviceucin was determined using NMR. Sviceucin displayed antimicrobial activity selectively against Gram-positive bacteria and inhibition of fsr quorum sensing in Enterococcus faecalis. This study adds sviceucin to the type I lasso peptide family as a new representative. Moreover, new clusters encoding disulfide-bond containing lasso peptides from Actinobacteria were identified by genome mining. Genetic and functional analyses revealed that the formation of disulfide bonds in sviceucin does not require a pathway-encoded thiol-disulfide oxidoreductase. Most importantly, we demonstrated the functional exchangeability of the sviceucin and microcin J25 (a non-disulfide-bridged lasso peptide) macrolactam synthetases in vitro, highlighting the potential of hybrid lasso synthetases in lasso peptide engineering.

The human immunodeficiency virus, type 1, Tat protein plays a key role in virus multiplication. Because of its apoptotic property, its production is highly controlled. It depends upon the A3 splicing site utilization. A key control of site A3 activity is the ESS2 splicing silencer, which is located within the long stem-loop structure 3 (SLS3), far downstream from site A3. Here, by enzymatic footprints, we demonstrate the presence of several heterogeneous nuclear ribonucleoprotein (hnRNP) A1-binding sites on SLS3 and show the importance of the C-terminal Gly domain of hnRNP A1 in the formation of stable complexes containing several hnRNP A1 molecules bound on SLS3. Mutations in each of the UAG triplets in ESS2 strongly reduce the overall hnRNP A1 binding, showing the central role of ESS2 in hnRNP A1 assembly on SLS2-SLS3. Using NMR spectroscopy, we demonstrate the direct interaction of ESS2 with the RNA recognition motifs domains of hnRNP A1. This interaction has limited effect on the RNA two-dimensional structure. The SR proteins SC35 and SRp40 were found previously to be strong activators of site A3 utilization. By enzymatic and chemical footprints, we delineate their respective binding sites on SLS2 and SLS3 and find a strong similarity between the hnRNP A1-, SC35-, and SRp40-binding sites. The strongest SC35-binding site only has a modest contribution to site A3 activation. Hence, the main role of SR proteins at site A3 is to counteract hnRNP A1 binding on ESS2 and ESE2. Indeed, we found that ESE2 has inhibitory properties because of its ability to bind hnRNP A1.

Splicing of the HIV-1 primary transcript is highly regulated. Maintenance of proper equilibrium among spliced, unspliced and partially spliced transcripts is essential for replication of the virus. Interaction between an RNA sequence within tat exon 2, called the proximal exonic splicing silencer two (pESS2), and its binding protein, the heterogeneous nuclear ribonucleoprotein H (hnRNP H), is crucial for the splicing regulation of the upstream intron. A preliminary structural study of the two biomolecules forming the complex is presented. The biomolecules design strategy chosen to make the analysis feasible by NMR and their production is presented. The first NMR data establishing the RNA secondary structure is shown, along with the preliminary NMR results obtained with the protein constructs. They found the feasibility of the structural project. To cite this article: S. Cabal, E. Guittet, C. R. Chimie 9 (2006).

Many pathogenic viruses use a programmed -1 translational frameshifting mechanism to regulate synthesis of their structural and enzymatic proteins. Frameshifting is vital for viral replication. A slippery sequence bound at the ribosomal A and P sites as well as a downstream stimulatory RNA structure are essential for frameshifting. Conflicting data have been reported concerning the structure of the downstream RNA signal in human immunodeficiency virus type 1 (HIV-1). Here, the solution structure of the HIV-1 frameshifting RNA signal was solved by heteronuclear NMR spectroscopy. This structure reveals a long hairpin fold with an internal three-nucleotide bulge. The internal loop introduces a bend between the lower and upper helical regions, a structural feature often seen in frameshifting pseudoknots. The NMR structure correlates with chemical probing data. The upper stem rich in conserved G-C Watson-Crick base-pairs is highly stable, whereas the bulge region and the lower stem are more flexible.

The widespread beta-thymosin/WH2 actin binding domain has versatile regulatory properties in actin dynamics and motility. beta-thymosins (isolated WH2 domain) maintain monomeric actin in a "sequestered" nonpolymerizable form. In contrast, when repeated in tandem or inserted in modular proteins, the beta-thymosin/WH2 domain promotes actin assembly at filament barbed ends, like profilin. The structural basis for these opposite functions is addressed using ciboulot, a three beta-thymosin repeat protein. Only the first repeat binds actin and possesses the function of ciboulot. The region that shows the strongest interaction with actin is an amphipathic N-terminal alpha helix, present in all beta-thymosin/WH2 domains, which recognizes the ATP bound actin structure and uses the shear motion of actin linked to ATP hydrolysis to control polymerization. Crystallographic ((1)H, (15)N), NMR, and mutagenetic data reveal that the weaker interaction of the C-terminal region of beta-thymosin/WH2 domain with actin accounts for the switch in function from inhibition to promotion of actin assembly.

A preliminary study of the dynamics of the domain 1 of annexin I by Nuclear Magnetic Resonance is presented. The use of 15N-relaxation-compensated CPMG experiments allowed us to show the presence of conformational motions in the micro–millisecond range that extend over several segments along the domain. For 14 residues, exchange rate constants could be evaluated, and were found in the range 1300–2500 s–1. To cite this article: M. Gallopin et al., C.R. Chimie 00 (2004).

In prokaryotes, the recoding of a UGA stop codon as a selenocysteine codon requires a special elongation factor (EF) SelB and a stem-loop structure within the mRNA called a selenocysteine insertion sequence (SECIS). Here, we used NMR spectroscopy to determine the solution structure of the SECIS mRNA hairpin and characterized its interaction with the mRNA-binding domain of SelB. Our structural and biochemical data identified the conserved structural features important for binding to EF SelB within different SECIS RNA sequences. In the free SECIS mRNA structure, conserved nucleotides are strongly exposed for recognition by SelB. Binding of the C-terminal domain of SelB stabilizes the RNA secondary structure. In the protein–RNA complex, a Watson–Crick loop base-pair leaves a GpU sequence accessible for SelB recognition. This GpU sequence at the tip of the capping tetraloop and a bulge uracil five Watson–Crick base-pairs apart from the GpU are essential for interaction with SelB.

International audience; In eukaryotes, mitochondrial iron-sulfur cluster (ISC), export and cytosolic iron-sulfur cluster assembly (CIA) machineries carry out biogenesis of iron-sulfur (Fe-S) clusters, which are critical for multiple essential cellular pathways. However, little is known about their export out of mitochondria. Here we show that Fe-S assembly of mitoNEET, the first identified Fe-S protein anchored in the mitochondrial outer membrane, strictly depends on ISC machineries and not on the CIA or CIAPIN1. We identify a dedicated ISC/export pathway in which augmenter of liver regeneration, a mitochondrial Mia40-dependent protein, is specific to mitoNEET maturation. When inserted, the Fe-S cluster confers mitoNEET folding and stability in vitro and in vivo. The holo-form of mitoNEET is resistant to NO and H2O2 and is capable of repairing oxidatively damaged Fe-S of iron regulatory protein 1 (IRP1), a master regulator of cellular iron that has recently been involved in the mitochondrial iron supply. Therefore, our findings point to IRP1 as the missing link to explain the function of mitoNEET in the control of mitochondrial iron homeostasis.

International audience; Solution-state nuclear magnetic resonance studies of membrane proteins are facilitated by the increased stability that trapping with amphipols confers to most of them as compared to detergent solutions. They have yielded information on the state of folding of the proteins, their areas of contact with the polymer, their dynamics, water accessibility, and the structure of protein-bound ligands. They benefit from the diversification of amphipol chemical structures and the availability of deuterated amphipols. The advantages and constraints of working with amphipols are discussed and compared to those associated with other non-conventional environments, such as bicelles and nanodiscs.

International audience; Nuclear magnetic resonance spectroscopy is a powerful tool to study structural and functional properties of proteins, provided that they can be enriched in stable isotopes such as (15)N, (13)C and (2)H. This is usually easy and inexpensive when the proteins are expressed in Escherichiacoli, but many eukaryotic (human in particular) proteins cannot be produced this way. An alternative is to express them in insect cells. Labeled insect cell growth media are commercially available but at prohibitive prices, limiting the NMR studies to only a subset of biologically important proteins. Non-commercial solutions from academic institutions have been proposed, but none of them is really satisfying. We have developed a (15)N-labeling procedure based on the use of a commercial medium depleted of all amino acids and supplemented with a (15)N-labeled yeast autolysate for a total cost about five times lower than that of the currently available solutions. We have applied our procedure to the production of a non-polymerizable mutant of actin in Sf9 cells and of fragments of eukaryotic and viral membrane fusion proteins in S2 cells, which typically cannot be produced in E. coli, with production yields comparable to those obtained with standard commercial media. Our results support, in particular, the putative limits of a self-folding domain within a viral glycoprotein of unknown structure.

Odorant-binding proteins (OBPs) are soluble, low-molecular-weight proteins secreted in the sensillum lymph surrounding the dendrites of olfactory sensilla from a wide range of insect species. These proteins play a role in the solubilization, transport and/or deactivation of pheromones and odorants. In order to study the relationships between the molecular structure in solution and their ligand-binding properties, we have (13)C/(15)N-double-labeled two divergent honeybee OBPs, called ASP1 and ASP2, in sufficient quantities to permit a full determination of the structure and dynamics using heteronuclear NMR spectroscopy. The recombinant labeled proteins produced by the methylotrophic yeast Pichia pastoris have been secreted into a buffered minimal medium using native insect signal peptide. Mass spectrometry and Edman sequencing showed a native-like processing with a labeling efficiency of secreted proteins greater than 98%. After dialysis, the recombinant proteins were purified to homogeneity by one-step reversed-phase liquid chromatography. The final yield after 4-day shake-flask liquid culture was approximately 60 and 100 mg/L for ASP1 and ASP2, respectively. The inexpensive overproduction of labeled recombinant ASP1 and ASP2 should allow NMR studies of the structures and ligand-binding analysis in order to understand the relationships between structure and biological function of these proteins.

DNA binding of the ethanol regulon transcription factor AlcR from Aspergillus nidulans was shown to involve a consensus basic region as in the other zinc cluster proteins. However, additional interactions between some residues and DNA were suspected, among which were a hypothetic hydrophobic interaction between Trp45 and the T residue of the consensus TGCGG sequence. In the present study, the differential chemical labeling of both the free protein and the protein/DNA complex showed significantly different behaviors of the three tryptophan residues comprised in the AlcR sequence toward the Koshland reagent. The spectacular decreased reaction rate for Trp45 within the complex confirmed the location of this residue at the protein/DNA interface. A similar result obtained with Trp53, an amino acid present at the C-terminal side of AlcR, also indicated its involvement in the DNA recognition. In contrast, the formation of the complex accompanied by an allosteric rearrangement allowed the Trp36 to be much more exposed to the solvent than in the free protein. These data provide additional evidence that the unique specificity of AlcR among the zinc binuclear cluster family results in new types of interactions between AlcR and its cognate targets. From a methodological point of view, the approach of differential chemical labeling combined with mass spectrometric analyses proved to be an interesting tool for the recognition of hydrophobic interactions between the tryptophan residues of a protein and its macromolecular target.

The isolated D2 domain of annexin I is unable to adopt a tertiary fold but exhibits both native and non-native residual structures. It thus constitutes an attractive model for the investigation of dynamics of partially folded states in the context of protein folding and stability. 15N relaxation parameters of the D2 domain have been acquired at three different magnetic fields, 500, 600 and 800 MHz. This enables the estimation of the contribution of conformational exchange to the relaxation parameters on the micro- to millisecond time scale, thus providing a suitable data set for the description of motions on the pico- and nanosecond time scale. The analysis of the seven spectral densities obtained (J(0), J(50 MHz), J(60 MHz), J(80 MHz), , , ) provides complementary and meaningful results on the conformational features of the D2 domain structure previously depicted by chemical shift and NOE data. Especially, residual helix segments exhibit distinct dynamical behaviors that are related to their intrinsic helical propensity. Beside the spectral density analysis, a series of models derived from the Lipari and Szabo model-free approach are investigated. Two models containing three parameters are able to reproduce equally well the experimental data within experimental errors but provide different values of order parameters and correlation times. The inability to find a unique model to describe the data emphasizes the difficulty to use and interpret the model-free parameters in the case of partially or fully unfolded proteins consisting of a wide range of interconverting conformers.

Aminoacyl-tRNA synthetases of higher eukaryotes possess polypeptide extensions in contrast to their prokaryotic counterparts. These extra domains of poorly understood function are believed to be involved in protein–protein or protein–RNA interactions. Here we showed by gel retardation and filter binding experiments that the repeated units that build the linker region of the bifunctional glutamyl-prolyl-tRNA synthetase had a general RNA-binding capacity. The solution structure of one of these repeated motifs was also solved by NMR spectroscopy. One repeat is built around an antiparallel coiled-coil. Strikingly, the conserved lysine and arginine residues form a basic patch on one side of the structure, presenting a suitable docking surface for nucleic acids. Therefore, this repeated motif may represent a novel type of general RNA-binding domain appended to eukaryotic aminoacyl-tRNA synthetases to serve as a cis-acting tRNA-binding cofactor.

Aspergillus nidulans est un champignon filamenteux capable d'utiliser l'ethanol comme source unique d'energie. La proteine AlcR est responsable de l'activation de l'expression des genes du regulon ethanol. Le domaine de liaison a l'ADN est situe dans la partie N-terminale de la proteine (a.a. 1 a 60), et sa structure determinee par RMN en solution montre un repliement global en bouquet binucleaire a zinc, avec deux helices supplementaires par rapport au motif de base. Alors que les structures deja connues de complexes ADN - bouquets binucleaires permettent de situer dans le grand sillon la quasi-totalite des interactions, nous montrons dans la presente etude l'implication du debut de la sequence dans la reconnaissance du petit sillon de l'ADN (a.a. 5 et 6).

A new pH-dependent off-resonance ROESY-HSQC experiment has been used to characterize the degree of protection of the amide protons of cryptogein, a protein of the elicitin family, against solvent exchange. The study of the pH dependence of solvent-shielded amide protons in this protein reveals that the helices have different levels of stability. Two of the five helices exhibit strong protection of amide hydrogens against exchange with the solvent. By contrast, greater flexibility is observed in the other three helices, particularly in the C-terminal helix. These results provide information on the dynamic features of the protein and are consistent with the RMSD for the backbone atoms of residues involved in helical structures. In addition, the question of the flexibility in a hydrophobic cavity made of conserved residues, which represent a plausible binding site, is addressed by this method.

The enzyme glucosamine-6P Synthase (Gfat, l -glutamine: d -fructose-6P amidotransferase) is involved in the hexosamine biosynthetic pathway and catalyzes the formation of glucosamine-6P from the substrates d -fructose-6-phosphate and l -glutamine. In eukaryotic cells, Gfat is inhibited by UDPGlcNAc, the end product of the biochemical pathway. In this work we present the dissection of the binding and inhibition properties of this feedback inhibitor and of its fragments by a combination of STD-NMR experiments and inhibition measurements on the wild type human enzyme (hGfat) as well as on site-directed mutants. We demonstrate that the UDPGlcNAc binding site is located in the isomerase domain of hGfat. Two amino acid residues (G445 and G461) located at the bottom of the binding site are identified to play a key role in the specificity of UDPGlcNAc inhibition of hGfat activity vs its bacterial Escherichia coli counterpart. We also show that UDPGlcNAc subcomponents have distinct features: the nucleotidic moiety is entirely responsible for binding whereas the N -acetyl group is mandatory for inhibition but not for binding, and the sugar moiety acts as a linker between the nucleotidic and N -acetyl groups. Combining these structural recognition determinants therefore appears as a promising strategy to selectively inhibit hGfat, which may for example help reduce complications in diabetes.

The structure of the second major adduct formed by the antitumor drug cisplatin with DNA, the intrastand cis–Pt(NH3)2{d(ApG)N7–N7} chelate (A*G*), has been investigated using a double-stranded nonanucleotide, d(CTCA*G*CCTC)-d(GAGGCTGAG), by means of NMR and molecular modeling. The NMR data allow us to conclude that the oligonucleotide is kinked at the platinated site towards the major groove in a way similar to that observed elsewhere for the G*G*-crosslink in d(GCCG*G*ATCGC)-d(GCGATCCGGC). The main difference concerns the position of the thymine T(15) complementary to the platinated adenine A*(4). It remains stacked on its 5′-neighbor C(14), corresponding to the "model E" described previously, whereas in the G*G*-adduct, the cytosine facing the 5′-G* was found to oscillate between the 5′-branch ("model E") and the 3′-branch ("model C") of the complementary strand. Two "E-type" models are presented which account for the particular NOE connectivity and for two remarkable upfield NMR signals: those of the H2′ proton of the cytidine C(3) 5′ to the A*G* chelate, and of the H3 imino proton of T(15), the base complementary to A*(4). The former shift is attributed to shielding by the destacked A*(4) base, whereas the latter is accounted for by a swinging movement of the T(15) base between two positions where the imino Watson-Crick hydrogen bond with A*(4) remains intact and the amino hydrogen bond is disrupted, or vice versa. Possible implications of the structural difference between the AG and GG adducts of cisplatin in the mutagenic properties of the two adducts are discussed.

BOP-Cl was found to be an efficient coupling reagent for the introduction of thiopeptide bonds on imino acid residues (Pro, Sar). Boc-amino monothioacids were coupled at moderate temperature (0 degree C-RT) with fair yields and with retained optical purity. The mechanism of the coupling reaction is discussed.

International audience; Neuroglobin (Ngb) is a globin present in the brain and retina of mammals. This hexacoordinated hemoprotein binds small diatomic molecules, albeit with lower affinity compared with other globins. Another distinctive feature of most mammalian Ngb is their ability to form an internal disulfide bridge that increases ligand affinity. As often seen for prosthetic heme b containing proteins, human Ngb exhibits heme heterogeneity with two alternative heme orientations within the heme pocket. To date, no details are available on the impact of heme orientation on the binding properties of human Ngb and its interplay with the cysteine oxidation state. In this work, we used (1)H NMR spectroscopy to probe the cyanide binding properties of different Ngb species in solution, including wild-type Ngb and the single (C120S) and triple (C46G/C55S/C120S) mutants. We demonstrate that in the disulfide-containing wild-type protein cyanide ligation is fivefold faster for one of the two heme orientations (the A isomer) compared with the other isomer, which is attributed to the lower stability of the distal His64-iron bond and reduced steric hindrance at the bottom of the cavity for heme sliding in the A conformer. We also attribute the slower cyanide reactivity in the absence of a disulfide bridge to the tighter histidine-iron bond. More generally, enhanced internal mobility in the CD loop bearing the disulfide bridge hinders access of the ligand to heme iron by stabilizing the histidine-iron bond. The functional impact of heme disorder and cysteine oxidation state on the properties of the Ngb ligand is discussed.

Fourier transform infrared (FTIR), UV absorption and exchangeable proton NMR spectroscopies have been used to study the formation and stability of two intramolecular pH-dependent triple helices composed by a chimeric 29mer DNA-RNA (DNA double strand and RNA third strand) or by the analogous 29mer RNA. In both cases decrease of pH induces formation of a triple helical structure containing either rU*dA.dT and rC+*dG.dC or rU*rA.rU and rC+*rG.rC triplets. FTIR spectroscopy shows that exclusively N-type sugars are present in the triple helix formed by the 29mer RNA while both N- and S-type sugars are detected in the case of the chimeric 29mer DNA-RNA triple helix. Triple helix formation with the third strand RNA and the duplex as DNA appears to be associated with the conversion of the duplex part from a B-form secondary structure to one which contains partly A-form sugars. Thermal denaturation experiments followed by UV spectroscopy show that a major stabilization occurs upon formation of the triple helices. Monophasic melting curves indicate a simultaneous disruption of the Hoogsteen and Watson-Crick hydrogen bonds in the intramolecular triplexes when the temperature is increased. This is in agreement with imino proton NMR spectra recorded as a function of temperature. Comparison with experiments concerning intermolecular triplexes of identical base and sugar composition shows the important role played by the two tetrameric loops in the stabilization of the intramolecular triple helices studied.

Stereo and regrochemcal variations in conjugate addition reactions leading to isoarborinal and fernenol-Hke interpenes via a five-step protccol are described. The Gngnard addition on 9. , 10. , 15. , 18. served lo establish the relative stereoehemicul details.

International audience; Non-natural amino acids are important tools for site-selective probing of peptide properties and interactions. Here, for the first time a fluorescent l-amino acid, exhibiting excited-state intramolecular proton transfer (ESIPT) and hydration-sensitive dual emission, was synthesized. It is an analogue of l-tryptophan bearing a slightly larger 2-(2-furyl)-3-hydroxychromone aromatic moiety instead of indole. This new amino acid was incorporated through solid-phase synthesis into NC(11-55), the zinc finger domain of the HIV-1 nucleocapsid protein, that exhibits potent nucleic acid chaperone properties. It was substituted for the Trp37 and Ala30 residues, located in the distal finger motif and the linker between the fingers of NC(11-55), respectively. Though the highly conserved Trp37 residue plays a key role in NC(11-55) structure and activity, its substitution for the new fluorescent analogue preserved the folding, the nucleic acid binding and chaperone activity of the peptide, indicating that the new amino acid can conservatively substitute Trp residues. In the presence of oligonucleotides, the Trp37-substituted peptide, but not the Ala30 variant, showed strong changes of the dual emission corresponding to local dehydration. The results are in line with NMR data, suggesting that the fluorescent amino acid interacts similarly to Trp37 with the nucleobases and is thus screened from water. Due to the exceptional sensitivity of its ESIPT fluorophore to hydration in highly polar environment, the new amino acid appears as a promising tool for substituting Trp residues and site-selectively investigating peptide-nucleic acid complexes.

International audience; The symmetry of i-motif tetramers gives to cytidine-rich oligonucleotides the capacity to associate into supramolecular structures (sms). In order to determine how the tetramers are linked together in such structures, we have measured by gel filtration chromatography and NMR the formation and dissociation kinetics of sms built by oligonucleotides containing two short C stretches separated by a non-cytidine-base. We show that a stretch of only two cytidines either at the 3'- or 5'-end is long enough to link the tetramers into sms. The analysis of the properties of sms formed by oligonucleotides differing by the length of the oligo-C stretches, the sequence orientation and the nature of the non-C base provides a model of the junction connecting the tetramers in sms.

International audience; β-Thymosin (βT) and WH2 domains are widespread, intrinsically disordered actin-binding peptides that display significant sequence variability and different regulations of actin self-assembly in motile and morphogenetic processes. Here, we reveal the structural mechanisms by which, in their 1:1 stoichiometric complexes with actin, they either inhibit assembly by sequestering actin monomers like Thymosin-β4, or enhance motility by directing polarized filament assembly like Ciboulot βT. We combined mutational, functional or structural analysis by X-ray crystallography, SAXS (small angle X-ray scattering) and NMR on Thymosin-β4, Ciboulot, TetraThymosinβ and the long WH2 domain of WASP-interacting protein. The latter sequesters G-actin with the same molecular mechanisms as Thymosin-β4. Functionally different βT/WH2 domains differ by distinct dynamics of their C-terminal half interactions with G-actin pointed face. These C-terminal interaction dynamics are controlled by the strength of electrostatic interactions with G-actin. At physiological ionic strength, a single salt bridge with actin located next to their central LKKT/V motif induces G-actin sequestration in both isolated long βT and WH2 domains. The results open perspectives for elucidating the functions of βT/WH2 domains in other modular proteins.

A 29-base RNA oligomer has been chemically synthesized and shown to form an intramolecular triple helix in solution at acidic pH. Assignment of the majority of the exchangeable proton NMR resonances demonstrated the Watson—Crick and Hoogsteen base pairings consistent with folding to form pyrimidine—purine—pyrimidine base triplets. FTIR spectroscopy provided independent evidence of base triplet formation, and indicated a predominately C3′-endo sugar pucker. UV absorption as a function of temperature suggested monophasic melting behaviour, which was confirmed by NMR of the imino protons.

The 3D solution structure of α-cobratoxin, a neurotoxin purified from the Naja naja siamensis snake venom, has been determined by Nuclear Magnetic Resonance spectroscopy, in conjunction with distance geometry and restrained molecular dynamics, at pH 7.5. A total of 490 distance restraints were obtained from NOE intensities and 25 φ dihedral angle restraints deduced from J- coupling data. The generated structures are well defined with root mean square deviations from a geometrical mean structure of 0.107 ± 0.036 nm for the backbone atoms and 0.128 ±0.073 nm for the side-chain atoms (considering residues 1 to 66 minus 26 to 35). A comparison between the generated structures at pH 7.5 and the mean NMR solution structure at pH 3.2 revealed that the 3D structure of α-cobratoxin is more compact at neutral pH. This major difference is mainly due to the pH-dependant conformational variations of three residues His(18), Thr(44) and Thr(59).

Arylcycloalkanes are produced from ω-alkenyl benzylselenides on reaction with alkyllithiums or on Lewis acid mediated electrophilic cyclisation.

Les temps de relaxation T 1 et T 1p et les nOes heteronucleaires a l'etat stationnaire des spins N 15 ont ete mesures sur une proteine de 98 residus, la capsiceine. L'analyse classique de ces donnees, qui utilise directement le formalisme de Lipari et Szabo, conduit a des resultats incoherents, vraisemblablement dus a la presence d'un echange lent le long de toute la proteine qui rend impossible l'evaluation prealable du temps de correlation global de la capsiceine a partir de l'expression des densites spectrales donnees par Lipari et Szabo

International audience; The mechanism of signal transduction mediated by G protein-coupled receptors is a subject of intense research in pharmacological and structural biology. Ligand association to the receptor constitutes a critical event in the activation process. Solution-state NMR can be amenable to high-resolution structure determination of agonist molecules in their receptor-bound state by detecting dipolar interactions in a transferred mode, even with equilibrium dissociation constants below the micromolar range. This is possible in the case of an inherent ultra-fast diffusive association of charged ligands onto a highly charged extracellular surface, and by slowing down the (1)H-(1)H cross-relaxation by perdeuterating the receptor. Here, we demonstrate this for two fatty acid molecules in interaction with the leukotriene BLT2 receptor, for which both ligands display a submicromolar affinity.

International audience; Dengue virus (DENV) causes the major arboviral disease of the tropics, characterized in its severe forms by signs of hemorrhage and plasma leakage. DENV encodes a nonstructural glycoprotein, NS1, that associates with intracellular membranes and the cell surface. NS1 is eventually secreted as a soluble hexamer from DENV-infected cells and circulates in the bloodstream of infected patients. Extracellular NS1 has been shown to modulate the complement system and to enhance DENV infection, yet its structure and function remain essentially unknown. By combining cryoelectron microscopy analysis with a characterization of NS1 amphipathic properties, we show that the secreted NS1 hexamer forms a lipoprotein particle with an open-barrel protein shell and a prominent central channel rich in lipids. Biochemical and NMR analyses of the NS1 lipid cargo reveal the presence of triglycerides, bound at an equimolar ratio to the NS1 protomer, as well as cholesteryl esters and phospholipids, a composition evocative of the plasma lipoproteins involved in vascular homeostasis. This study suggests that DENV NS1, by mimicking or hijacking lipid metabolic pathways, contributes to endothelium dysfunction, a key feature of severe dengue disease.

International audience; MED25 (ARC92/ACID1) is a 747 residues subunit specific to higher eukaryote Mediator complex, an essential component of the RNA polymerase II general transcriptional machinery. MED25 is a target of the Herpes simplex virus transactivator protein VP16. MED25 interacts with VP16 through a central MED25 PTOV (Prostate tumour overexpressed)/ACID (Activator interacting domain) domain of unknown structure. As a first step towards understanding the mechanism of recruitment of transactivation domains by MED25, we report here the NMR structure of the MED25 ACID domain. The domain architecture consists of a closed β-barrel with seven strands (Β1-Β7) and three α-helices (H1-H3), an architecture showing similarities to that of the SPOC (Spen paralog and ortholog C-terminal domain) domain-like superfamily. Preliminary NMR chemical shift mapping showed that VP16 H2 (VP16C) interacts with MED25 ACID through one face of the β-barrel, defined by strands B4-B7-B6.

Cyathoviridine 2 was isolated from the bark of Cyathostemma viridiflorum Griff. and its structure elucidated by 2D NMR analysis. 2 showed cytotoxicity against KB cells (IC50 4.5 μ/ml).