Backbone chemical shift assignments of human 14-3-3σ\sigma

14-3-3 proteins are a group of seven dimeric adapter proteins that exert their biological function by interacting with hundreds of phosphorylated proteins, thus influencing their sub-cellular localization, activity or stability in the cell. Due to this remarkable interaction network, 14-3-3 proteins have been associated with several pathologies and the protein-protein interactions established with a number of partners are now considered promising drug targets. The activity of 14-3-3 proteins is often isoform specific and to our knowledge only one out of seven isoforms, 14-3-3$\zeta$, has been assigned. Despite the availability of the crystal structures of all seven isoforms of 14-3-3, the additional NMR assignments of 14-3-3 proteins are important for both biological mechanism studies and chemical biology approaches. Herein, we present a robust backbone assignment of 14-3-3$\sigma$, which will allow advances in the discovery of potential therapeutic compounds. This assignment is now being applied to the discovery of both inhibitors and stabilizers of 14-3-3 protein-protein interactions

Phosphorylation and O-GlcNAcylation of the PHF-1 Epitope of Tau Protein Induce Local Conformational Changes of the C-Terminus and Modulate Tau Self-Assembly Into Fibrillar Aggregates

Phosphorylation of the neuronal microtubule-associated Tau protein plays a critical role in the aggregation process leading to the formation of insoluble intraneuronal fibrils within Alzheimer’s disease (AD) brains. In recent years, other posttranslational modifications (PTMs) have been highlighted in the regulation of Tau (dys)functions. Among these PTMs, the O-β-linked N-acetylglucosaminylation (O-GlcNAcylation) modulates Tau phosphorylation and aggregation. We here focus on the role of the PHF-1 phospho-epitope of Tau C-terminal domain that is hyperphosphorylated in AD (at pS396/pS404) and encompasses S400 as the major O-GlcNAc site of Tau while two additional O-GlcNAc sites were found in the extreme C-terminus at S412 and S413. Using high resolution NMR spectroscopy, we showed that the O-GlcNAc glycosylation reduces phosphorylation of PHF-1 epitope by GSK3β alone or after priming by CDK2/cyclin A. Furthermore, investigations of the impact of PTMs on local conformation performed in small peptides highlight the role of S404 phosphorylation in inducing helical propensity in the region downstream pS404 that is exacerbated by other phosphorylations of PHF-1 epitope at S396 and S400, or O-GlcNAcylation of S400. Finally, the role of phosphorylation and O-GlcNAcylation of PHF-1 epitope was probed in in-vitro fibrillization assays in which O-GlcNAcylation slows down the rate of fibrillar assembly while GSK3β phosphorylation stimulates aggregation counteracting the effect of glycosylation.Peer Reviewe

Structural Basis of Tau Interaction With BIN1 and Regulation by Tau Phosphorylation

Bridging integrator-1 (BIN1) gene is associated with an increased risk to develop Alzheimer’s disease, a tauopathy characterized by intra-neuronal accumulation of phosphorylated Tau protein as paired helical filaments. Direct interaction of BIN1 and Tau proteins was demonstrated to be mediated through BIN1 SH3 C-terminal domain and Tau (210–240) peptide within Tau proline-rich domain. We previously showed that BIN1 SH3 interaction with Tau is decreased by phosphorylation within Tau proline-rich domain, of at least T231. In addition, the BIN1/Tau interaction is characterized by a dynamic equilibrium between a closed and open conformations of BIN1 isoform 1, involving an intramolecular interaction with its C-terminal BIN1 SH3 domain. However, the role of the BIN1/Tau interaction, and its potential dysregulation in Alzheimer’s disease, is not yet fully understood. Here we showed that within Tau (210–240) peptide, among the two proline-rich motifs potentially recognized by SH3 domains, only motif P216TPPTR221 is bound by BIN1 SH3. A structural model of the complex between BIN1 SH3 and Tau peptide (213–229), based on nuclear magnetic resonance spectroscopy data, revealed the molecular detail of the interaction. P216 and P219 within the proline-rich motif were in direct contact with the aromatic F588 and W562 of the BIN1 SH3 domain. The contact surface is extended through electrostatic interactions between the positively charged R221 and K224 residues of Tau peptide and those negatively charged of BIN1 SH3, corresponding to E556 and E557. We next investigated the impact of multiple Tau phosphorylations within Tau (210–240) on its interaction with BIN1 isoform 1. Tau (210–240) phosphorylated at four different sites (T212, T217, T231, and S235), contrary to unphosphorylated Tau, was unable to compete with the intramolecular interaction of BIN1 SH3 domain with its CLAP domain. In accordance, the affinity of BIN1 SH3 for phosphorylated Tau (210–240) peptide was reduced, with a five-fold increase in the dissociation constant, from a Kd of 44 to 256 μM. This study highlights the complexity of the regulation of BIN1 isoform 1 with Tau. As abnormal phosphorylation of Tau is linked to the pathology development, this regulation by phosphorylation might have important functional consequences

Making 1H-1H couplings more accessible and accurate with selective 2DJ NMR experiments aided by 13C satellites

1H-1H coupling constants are one of the primary sources of information for NMR structural analysis. Several selective 2DJ experiments have been proposed that allow their individual measurement at pure shift resolution. However, all these experiments fail in the not uncommon case when coupled protons have very close chemical shifts. Firstly, the coupling between protons with overlapping multiplets is inaccessible due to the inability of a frequency-selective pulse to invert just one of them. Secondly, the strong coupling condition affects the accuracy of coupling measurements involving third spins. These shortcomings impose a limit on the effectiveness of state-of-the-art experiments, such as G-SERF or PSYCHEDELIC. Here, we introduce two new and complementary selective 2DJ experiments that we coin SERFBIRD and SATASERF. These experiments overcome the aforementioned issues by utilizing the 13C satellite signals at natural isotope abundance, which resolve the chemical shift degeneracy. We demonstrate the utility of these experiments on the tetrasaccharide stachyose and the challenging case of norcamphor, for the latter achieving measurement of all JHH couplings while only few were accessible with PSYCHEDELIC. The new experiments are applicable to any organic compound and will prove valuable for configurational and conformational analyses

Interaction study between HCV NS5A-D2 and NS5B using 19F NMR

The non structural protein 5A (NS5A) regulates the replication of the hepatitis C viral RNA through a direct molecular interaction of its domain 2 (NS5A-D2) with the RNA dependent RNA polymerase NS5B. Because of conflicting data in the literature, we study here this molecular interaction using fluorinated versions of the NS5A-D2 protein derived from the JFH1 Hepatitis C Virus strain. Two methods to prepare fluorine-labelled NS5A-D2 involving the biosynthetic incorporation of a F-19-tryptophan using 5-fluoroindole and the posttranslational introduction of fluorine by chemical conjugation of 2-iodo-N-(trifluoromethyl)acetamide with the NS5A-D2 cysteine side chains are presented. The dissociation constants (K-D) between NS5A-D2 and NS5B obtained with these two methods are in good agreement, and yield values comparable to those derived previously from a surface plasmon resonance study. We compare benefits and limitations of both labeling methods to study the interaction between an intrinsically disordered protein and a large molecular target by F-19 NMR

Making 1H– 1H Couplings More Accessible and Accurate with Selective 2DJ NMR Experiments Aided by 13C Satellites

International audience1H-1H coupling constants are one of the primary sources of information for nuclear magnetic resonance (NMR) structural analysis. Several selective 2DJ experiments have been proposed that allow for their individual measurement at pure shift resolution. However, all of these experiments fail in the not uncommon case when coupled protons have very close chemical shifts. First, the coupling between protons with overlapping multiplets is inaccessible due to the inability of a frequency-selective pulse to invert just one of them. Second, the strong coupling condition affects the accuracy of coupling measurements involving third spins. These shortcomings impose a limit on the effectiveness of state-of-the-art experiments, such as G-SERF or PSYCHEDELIC. Here, we introduce two new and complementary selective 2DJ experiments that we coin SERFBIRD and SATASERF. These experiments overcome the aforementioned issues by utilizing the 13C satellite signals at natural isotope abundance, which resolves the chemical shift degeneracy. We demonstrate the utility of these experiments on the tetrasaccharide stachyose and the challenging case of norcamphor, for the latter achieving measurement of all J HH couplings, while only a few were accessible with PSYCHEDELIC. The new experiments are applicable to any organic compound and will prove valuable for configurational and conformational analyses

Etude par RMN de protéines et de peptides régulateurs de la poymérisation de l'actine

Les protéines à domaine WH2/thymosine b, notamment la thymosine b4 et le domaine l de ciboulot, jouent un rôle important dans le mécanisme de régulation de la polymérisation de l actine. La thymosine b4 inhibe la formation des filaments de l'actine par la séquestration des monomères. A l'inverse, ciboulot D1 participe à la polymérisation de l'actine. Une série de protéines chimères de la thymosine b4 et du premier domaine de ciboulot ont été construites et étudiées afin d'évaluer l'influence de chaque région des domaines WH2/Tb (N-terminale, linker, motif consensus et C-terminale) sur leurs interactions avec l'actine et d'identifier les déterminants des fonctions séquestrantes ou promotrices de la formation du filament. Les complexes des protéines à domaine WH2/Tb avec l'actine ont été caractérisés de manière détaillée d'un point de vue structural et dynamique. Ces études ont permis d'affiner le modèle fonctionnel, de mettre en évidence des éléments importants de l'interaction de ces protéines avec l'actine et d'analyser le comportement des différentes protéines selon leur fonction. L'influence de peptides vecteurs amphiphiles et/ou chargés positivement sur la polymérisation de l'actine a été abordée par RMN. Leur liaison au monomère d'actine et la formation induite de particules de haute masse ont pu être mises en évidence. En parallèle d'expériences de couplage chimique, le déplacement de la thymosine 4 a permis de proposer un site d'interaction pour ces peptides. Ces peptides ne peuvent donc plus être considérés comme de simples vecteurs passifs et leur rôle propre dans la restauration du filament ouvre d'intéressantes perspectives.bthymosin and WH2 domains are widespread, intrinsically disordered actin-binding peptides which display versatile regulation of actin assembly in motile processes. Thymosin-b4 sequesters G-actin, whereas the complex of G-actin with the first b-thymosin domain of Ciboulot or conventional WH2 domains participates in filament barbed end assembly like profilin. Here, using chimeras of ciboulot and Thymosin-b4, we show the role of their different regions for their binding and function with actin and the regulation of the dynamics in tbeir 1:1 complexes with G-actin at physiological ionic strength. A single salt bridge with actin located in Thymosin-b4 linker before its LKKT/V motif stabilizes aIl its central and C-terminal interactions and induces sequestration when inserted in Ciboulot. NMR relaxation structural studies are combined on CibD1/T b 4 chimeras to show that the central and C-terminal regions of cibD1 and Tb4 display an equivalent G-actin sequestering capacity and that the sequestering and profilin-like activities of both bT and long WH2 dornains are regulated at physiological ionic strength by dynamic interactions in their stoichiometric 1:1 complexes with G-actin. Cell-penetrating peptides can cross cell membranes and are commonly seen as biologically inert molecules. However, we found that some CPP could remodel actin cytoskeleton. These arginine- and/or tryptophan-rich peptides could cross cell membrane and induced stress fibers formation. The peptides could bind directly monomeric actin as determined by NMR and calorimetry studies. Therefore, cell-penetrating peptides might interact with intracellular protein partners, such as actin.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Hepatitis E Virus RNA‐Dependent RNA Polymerase is Involved in RNA Replication and Infectious Particle Production

International audienceBackground and Aims: Hepatitis E virus (HEV) is one of the most common causes of acute hepatitis worldwide. Its positive-strand RNA genome encodes three open reading frames (ORF). ORF1 is translated into a large protein composed of multiple domains and known as the viral replicase. The RNA-dependent RNA polymerase (RdRp) domain is responsible for the synthesis of viral RNA.Approach and Results: Here, we identified a highly conserved α-helix located in the RdRp thumb subdomain. Nuclear magnetic resonance demonstrated an amphipathic α-helix extending from amino acids 1628 to 1644 of the ORF1 protein. Functional analyses revealed a dual role of this helix in HEV RNA replication and virus production, including assembly and release. Mutations on the hydrophobic side of the amphipathic α-helix impaired RNA replication and resulted in the selection of a second-site compensatory change in the RdRp palm subdomain. Other mutations enhanced RNA replication but impaired virus assembly and/or release.Conclusions: Structure-function analyses identified a conserved amphipathic α-helix in the thumb subdomain of the HEV RdRp with a dual role in viral RNA replication and infectious particle production. This study provides structural insights into a key segment of the ORF1 protein and describes the successful use of reverse genetics in HEV, revealing functional interactions between the RdRp thumb and palm subdomains. On a broader scale, it demonstrates that the HEV replicase, similar to those of other positive-strand RNA viruses, is also involved in virus productio

1H, 13C, and 15N chemical shift assignment of human PACSIN1/syndapin I SH3 domain in solution

International audienceHuman neuron-specific PACSIN1 plays a key role in synaptic vesicle recycling and endocytosis, as well as reorganization of the microtubule dynamics to maintain axonal plasticity. PACSIN1 contains a highly conserved C-terminal SH3 domain and an F-bar domain at its N-terminus. Due to its remarkable interaction network, PACSIN1 plays a central role in key neuronal functions. Here, we present a robust backbone and side-chain assignment of PACSIN1 SH3 domain based on 2D [1H,15N] HSQC or HMQC, and 3D BEST-HNCO, -HNCACB, -HN(CO)CACB, -HN(CA)CO, and standard (H)CC(CO)NH, HN(CA)NNH, HN(COCA)NH, HBHANNH, HNHA, HBHA(CO)NH, H(CC)(CO)NH, HCCH-TOCSY, that covers 96% for all 13CO, 13Cα and 13Cβ, 28% of 13Cγδε, and 95% of 1HN and 15N chemical shifts. Modelling based on sequence homology with a known related structure, and chemical shift-based secondary structure predictions, identified the presence of five β-strands linked by flexible loops. Taken together, these results open up new avenues to investigate and develop new therapeutic strategies