The unusual structure of the PiggyMac cysteine-rich domain reveals zinc finger diversity in PiggyBac-related transposases.

BACKGROUND: Transposons 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 ciliate Paramecium tetraurelia is 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. RESULTS: We 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 from Trichoplusia ni. Instead, the arrangement of cysteines and histidines in the primary sequence of the Pgm CRD resembles that of active transposases from piggyBac-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. CONCLUSIONS: The 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 from Paramecium and Tetrahymena, was already present in the ancestral active transposase that gave rise to ciliate domesticated proteins.

NMR Characterization of the Influence of Zinc(II) Ions on the Structural and Dynamic Behavior of the New Delhi Metallo-ß-Lactamase-1 and on the Binding with Flavonols as Inhibitors.

New Delhi metallo-ß-lactamase-1 (NDM-1) has recently emerged as a global threat because of its ability to confer resistance to all common ß-lactam antibiotics. Understanding the molecular basis of ß-lactam hydrolysis by NDM is crucial for designing NDM inhibitors or ß-lactams resistant to their hydrolysis. In this study, for the first time, NMR was used to study the influence of Zn(II) ions on the dynamic behavior of NDM-1. Our results highlighted that the binding of Zn(II) in the NDM-1 active site induced several structural and dynamic changes on active site loop 2 (ASL2) and L9 loops and on helix α2. We subsequently studied the interaction of several flavonols: morin, quercetin, and myricetin were identified as natural and specific inhibitors of NDM-1. Quercetin conjugates were also synthesized in an attempt to increase the solubility and bioavailability. Our NMR investigations on NDM-1/flavonol interactions highlighted that both Zn(II) ions and the residues of the NDM-1 ASL1, ASL2, and ASL4 loops are involved in the binding of flavonols. This is the first NMR interaction study of NDM-1/inhibitors, and the models generated using HADDOCK will be useful for the rational design of more active inhibitors, directed against NDM-1.

Sequence-specific DNA binding activity of the cross-brace zinc finger motif of the piggyBac transposase.

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.

Functional Modulation of a G Protein-Coupled Receptor Conformational Landscape in a Lipid Bilayer.

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.

Initial Molecular Recognition Steps of McjA Precursor during Microcin J25 Lasso Peptide Maturation.

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.

Anacardic Acids from Knema hookeriana as Modulators of Bcl-xL/Bak and Mcl-1/Bid Interactions.

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 Ki values ranging from 0.2 to 18 µM. 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.

Redox Control of the Human Iron-Sulfur Repair Protein MitoNEET Activity via Its Iron-Sulfur Cluster.

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.

Characterization of Sviceucin from Streptomyces Provides Insight into Enzyme Exchangeability and Disulfide Bond Formation in Lasso Peptides.

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.

A well-balanced preexisting equilibrium governs electron flux efficiency of a multidomain diflavin reductase.

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.

Robust and low cost uniform (15)N-labeling of proteins expressed in Drosophila S2 cells and Spodoptera frugiperda Sf9 cells for NMR applications.

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.

The diabetes drug target MitoNEET governs a novel trafficking pathway to rebuild an Fe-S cluster into cytosolic aconitase/iron regulatory protein 1.

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.

The use of amphipols for solution NMR studies of membrane proteins: advantages and constraints as compared to other solubilizing media.

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.

Mapping the UDP-N-acetylglucosamine regulatory site of human glucosamine-6P synthase by saturation-transfer difference NMR and site-directed mutagenesis.

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.

(1)H, (13)C and (15)N NMR assignment of CGC-19, a single domain proteic constituent of a non ribosomal peptide synthetase.

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.

Heme orientation modulates histidine dissociation and ligand binding kinetics in the hexacoordinated human neuroglobin.

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.

Two-color fluorescent l-amino acid mimic of tryptophan for probing peptide-nucleic acid complexes.

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.

The closed and compact domain organization of the 70-kDa human cytochrome P450 reductase in its oxidized state as revealed by NMR.

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 (15)N relaxation and (1)H-(15)N 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.

Junctions between i-motif tetramers in supramolecular structures.

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.

Sequence determinants governing the topology and biological activity of a lasso peptide, microcin J25.

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.

How a single residue in individual ß-thymosin/WH2 domains controls their functions in actin assembly.

ß-Thymosin (ßT) 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-ß4, or enhance motility by directing polarized filament assembly like Ciboulot ßT. We combined mutational, functional or structural analysis by X-ray crystallography, SAXS (small angle X-ray scattering) and NMR on Thymosin-ß4, Ciboulot, TetraThymosinß and the long WH2 domain of WASP-interacting protein. The latter sequesters G-actin with the same molecular mechanisms as Thymosin-ß4. Functionally different ßT/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 ßT and WH2 domains. The results open perspectives for elucidating the functions of ßT/WH2 domains in other modular proteins.