Développement et application de méthodologies RMN à l'étude structurale et dynamique de macromolécules en solution

La caractérisation de la structure et de la dynamique de biomolécules par RMN représente un grand enjeu dans l étude de systèmes biologiques. Le développement de méthodes complémentaires à l étude classique en RMN, telles que l étude des fluctuations concertées des déplacements chimiques isotropes nous a permis de caractériser la présence de mouvements lents dans un système biologique pertinent : un complexe de centrine humaine avec un peptide cible. Par ailleurs, les phénomènes d interférence impliquant un centre paramagnétique constituent, aujourd hui, un outil puissant pour obtenir de nouvelles contraintes dans le cadre d études structurales de macromolécules biologiques. Cette approche a été appliquée au même système dans lequel un ion Ca2+ a été remplacé par un ion paramagnetique. Enfin, nous avons développé une nouvelle méthode grâce à laquelle nous avons pu mesurer des vitesses d échange de protons avec le solvant atteignant les 105 s-1. Nous avons pu ainsi déterminer les vitesses d échange du proton indole du tryptophane dans ses formes cationique, zwitterionique, et anionique.PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Structure, Dynamics and Thermodynamics of the Human Centrin 2/hSfi1 Complex

Centrin, an EF-hand calcium-binding protein, has been shown to be involved in the duplication of centrosomes, and Sfi1 (Suppressor of fermentation-induced loss of stress resistance protein 1) is one of its centrosomal targets. There are three isoforms of human centrin, but here we only considered centrin 2 (HsCen2). This protein has the ability to bind to any of the similar to 25 repeats of human Sfi1 (hSfi1) with more or less affinity. In this study, we mainly focused on the 17th repeat (R17-hSfi1-20), which presents the highest level of similarity with a well-studied 17-residue peptide (P17-XPC) from human xeroderma pigmentosum complementation group C protein, another centrin target for DNA repair. The only known structure of HsCen2 was resolved in complex with P17-XPC. The 20-residue peptide R17-hSfi1-20 exhibits the motif L8L4W1, which is the reverse of the XPC motif, W1L4L8. Consequently, the dipole of the helix formed by this motif has a reverse orientation. We wished to ascertain the impact of this reversal on the structure, dynamics and affinity of centrin. To address this question, we determined the structure of C-HsCen2 [the C-terminal domain of HsCen2 (T94-Y172)] in complex with R17-hSfi1-20 and monitored its dynamics by NMR, after having verified that the N-terminal domain of HsCen2 does not interact with the peptide The structure shows that the binding mode is similar to that of P17-XPC. However, we observed a 2 -angstrom translation of the R17-hSfi1-20 helix along its axis, inducing less anchorage in the protein and the disruption of a hydrogen bond between a tryptophan residue in the peptide and a well-conserved nearby glutamate in C+HsCen2. NMR dynamic studies of the complex strongly suggested the existence of an unusual calcium secondary binding mode in calcium-binding loop III made possible by the uncommon residue composition of this loop. The secondary metal site is only populated at high calcium concentration and depends on the type of bound ligand. (C) 2009 Elsevier Ltd. All rights reserve

Structural and functional analysis of the DEAF-1 and BS69 MYND domains.

DEAF-1 is an important transcriptional regulator that is required for embryonic development and is linked to clinical depression and suicidal behavior in humans. It comprises various structural domains, including a SAND domain that mediates DNA binding and a MYND domain, a cysteine-rich module organized in a Cys(4)-Cys(2)-His-Cys (C4-C2HC) tandem zinc binding motif. DEAF-1 transcription regulation activity is mediated through interactions with cofactors such as NCoR and SMRT. Despite the important biological role of the DEAF-1 protein, little is known regarding the structure and binding properties of its MYND domain.Here, we report the solution structure, dynamics and ligand binding of the human DEAF-1 MYND domain encompassing residues 501-544 determined by NMR spectroscopy. The structure adopts a ββα fold that exhibits tandem zinc-binding sites with a cross-brace topology, similar to the MYND domains in AML1/ETO and other proteins. We show that the DEAF-1 MYND domain binds to peptides derived from SMRT and NCoR corepressors. The binding surface mapped by NMR titrations is similar to the one previously reported for AML1/ETO. The ligand binding and molecular functions of the related BS69 MYND domain were studied based on a homology model and mutational analysis. Interestingly, the interaction between BS69 and its binding partners (viral and cellular proteins) seems to require distinct charged residues flanking the predicted MYND domain fold, suggesting a different binding mode. Our findings demonstrate that the MYND domain is a conserved zinc binding fold that plays important roles in transcriptional regulation by mediating distinct molecular interactions with viral and cellular proteins

The follicular fluid metabolome differs according to the endometriosis phenotype

International audienceResearch question: Is there a follicular fluid-specific metabolic profile in deep infiltrating endometriosis (DIE) depending on the presence of an associated ovarian endometrioma (OMA) that could lead to the identification of biomarkers for diagnosis and prognosis of the disease?Design: In this prospective cohort study, proton nuclear magnetic resonance (1H-NMR) experiments were carried out on 50 follicular fluid samples from patients presenting with DIE, associated or not associated with an OMA, and 29 follicular fluid samples from patients with infertility caused by a tubal obstruction.Results: Concentrations of glucose, citrate, creatine and amino acids such as tyrosine and alanine were lower in women with DIE than control participants, whereas concentrations of lactate, pyruvate, lipids and ketone bodies were higher. Metabolic analysis revealed enhanced concentrations of glycerol and ketone bodies in patients with OMA, indicative of an activation of lipolysis followed by beta-oxidation. Concentrations of lactate and pyruvate were increased in patients without OMA, whereas the concentration of glucose was decreased, highlighting activation of the anaerobic glycolysis pathway. Differences in concentrations of amino acids such as threonine and glutamine were also statistically relevant in discriminating between the presence or absence of OMA.Conclusions: Results indicate a mitochondrial dysregulation in endometriosis phenotypes, with a modified balance between anaerobic glycolysis and beta-oxidation in OMA phenotypes that could affect the fertility of women with endometriosis. As the composition of the follicular fluid has been shown to be correlated with oocyte development and outcome of implantation after fertilization, these findings may help explain the high level of infertility in these patients

Binding of DEAF-1 MYND to SMRT and NCoR peptides.

<p>(a) Chemical Shift Perturbations (CSP, see methods) observed for the interaction between DEAF-1 MYND domain and SMRT (top) and NCoR (bottom) corepressor peptides. The sequence of SMRT and NCoR peptides used for the titrations are indicated in each graph. Secondary structure elements and amino acid sequence of the DEAF-1 MYND domain are shown at the top of the panel. Residues expected to interact with the corepressors are colored magenta. CSPs of residues that are most strongly affected are shown (cross symbols) as a function of ligand concentration for both titration with SMRT (top right) and NCoR (bottom right). The observed CSPs were fitted to a binding isotherm yielding dissociation constanst of 5.30±0.54 mM and 3.08±0.12 mM for the SMRT and NCoR peptides, respectively. The binding curves are shown as dashed lines. (b) Superposition of ¹H, ¹⁵N HSQC spectra of a 1 mM sample of the free DEAF-1 MYND domain (red) and upon addition of unlabeled corepressor peptides (cyan) up to a final concentration of 8 mM and 5 mM of SMRT and NCoR peptide (1∶8 and 1∶5 molar ratio), respectively. The intermediate steps of each titration are zoomed for a sub-region of the corresponding spectrum. In either case binding takes place on the fast exchange regime with respect to the chemical shift time scale. (c) Ribbon representation of DEAF-1 (left) and ETO (right) MYND domains. Residues experiencing the largest chemical shift perturbation upon addition of the corepressor peptides are shown as magenta sticks. In the ETO-SMRT complex structure the corresponding residues are shown as sticks as well, and the SMRT ligand peptide is colored orange and shown in cartoon representation.</p

Primary sequence and NMR analysis of the DEAF-1 MYND domain.

<p>(a) Sequence alignment of different MYND domains. Residues coordinating the first and second zinc ions are highlighted with red and blue background, respectively. Residues involved in binding to corepressor peptides are indicated with a green star at the bottom, and those interacting through their side chains are highlighted in green. The positions of mutations performed on BS69 are indicated at the bottom. (b) Long range ¹H, ¹⁵N HSQC spectrum correlating H^ε1 and H^δ2 to N^δ1 and N^ε2 through ²<i>J</i>_HN and ³<i>J</i>_HN couplings (Pelton et al 1993). The spectrum reveals a different protonation pattern for each histidine sidechain corresponding to the three possible tautomeric states. (c) ¹³C secondary chemical shifts (top), {¹H}-¹⁵N heteronuclear NOE (middle), and ¹⁵N R₂/R₁ relaxation rates ratio (bottom) are plotted versus DEAF-1 MYND residue numbers. The secondary structure elements and the amino acid sequence of the protein are indicated at the top of the figure. Residues coordinating the first and second zinc are colored red and blue respectively.</p

Structural statistics of the DEAF-1 MYND domain.

<p>Statistics are given for the 20 lowest energy structures after water refinement out of 100 calculated.</p>1<p>Distance restraints were derived from NOE peak intensities using CYANA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054715#pone.0054715-Gntert1" target="_blank">[31]</a>, and then introduced as unambiguous distances in CNS. No distance restraint was violated by more than 0.5 Å.</p>2<p>Torsion angles were predicted using TALOS+ <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054715#pone.0054715-Shen1" target="_blank">[30]</a>. No dihedral angle restraint was violated by more than 5°.</p>3<p>RDC restraints were incorporated using a harmonic potential. Force constants of 0.2, 0.1, 0.3, and 0.6 kcal mol⁻¹ Hz⁻² for H^HN, NC′, H^NC′ and H^α C^α respectively, were used to reflect the estimated error in the measurement.</p>4<p>Ramachandran plot statistics were obtained using PROCHECK <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054715#pone.0054715-Laskowski1" target="_blank">[64]</a> for residues 502–541.</p

Binding studies and mutational analysis of BS69 MYND domain.

<p>(a) Two views of an electrostatic surface representation of the DEAF-1 MYND domain. Positive (blue) and negative (red) electrostatic surface potential is shown at ±3 k_B T/e⁻ and was determined using the program APBS <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054715#pone.0054715-Baker1" target="_blank">[66]</a> in Pymol (<a href="http://www.pymol.org" target="_blank">www.pymol.org</a>). (b) Corresponding surface views for the BS69 MYND structure obtained from homology modelling. Positive (blue) and negative (red) electrostatic surface potential is displayed at ±3 k_B T/e⁻. The location of the residues that were mutated for binding studies are labelled on the surface representation of the BS69 homology model. (c),(d) Analysis of binding of E1A, EBNA2 and MGA to wild-type or mutant BS69 proteins expressed as a GST-fusion protein or to the GST (G−) moiety as control; Inp: Input 10%; G-RRKR-559-562G4: mutation of residues 559–562 into four glycines. (e) A single point mutation in BS69 abrogates the binding to E1A. QT6 fibroblasts were transfected with 12SE1A and/or FLAG-tagged BS69 expression vectors as indicated. Cellular complexes were immunoprecipitated with an M2 anti-FLAG antibody. Co-immunoprecipitated and ectopic expressions of E1A were revealed by immunoblotting using an M73-E1A antibody.</p