1H, 13C and 15N NMR assignments of the E. coli peptide deformylase in complex with a natural inhibitor called actinonin

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Tether influence on the binding properties of tRNALys3 ligands designed by a fragment-based approach.

International audienceA small library of 1,5-triazole derivatives linking a diaminocyclopentadiol and aromatic ketones has been prepared and screened using NMR and fluorescent techniques against tRNA(Lys)(3), the HIV reverse transcription primer. The comparison of their binding properties to those of their 1,4-triazole isomers, previously discovered in a fragment-based approach, outlines the influence of the linker on affinity and binding selectivity in such an approach

RNA-methyltransferase TrmA is a dual-specific enzyme responsible for C 5 - methylation of uridine in both tmRNA and tRNA

International audienceIn bacteria, trans-translation rescues stalled ribosomes by the combined action of tmRNA (transfer-mRNA) and its associated protein SmpB. The tmRNA 5' and 3' ends fold into a tRNA-like domain (TLD), which shares structural and functional similarities with tRNAs. As in tRNAs, the UUC sequence of the T-arm of the TLD is post-transcriptionally modified to m ( 5) UψC. In tRNAs of gram-negative bacteria, formation of m ( 5) U is catalyzed by the SAM-dependent methyltransferase TrmA, while formation of m ( 5) U at two different positions in rRNA is catalyzed by distinct site-specific methyltransferases RlmC and RlmD. Here, we show that m ( 5) U formation in tmRNAs is exclusively due to TrmA and should be considered as a dual-specific enzyme. The evidence comes from the lack of m ( 5) U in purified tmRNA or TLD variants recovered from an Escherichia coli mutant strain deleted of the trmA gene. Detection of m ( 5) U in RNA was performed by NMR analysis

Co-expression of RNA–protein complexes in Escherichia coli and applications to RNA biology

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In vivo tmRNA protection by SmpB and pre-ribosome binding conformation in solution

International audienceTmRNA is an abundant RNA in bacteria with tRNA and mRNA features. It is specialized in trans-translation, a translation rescuing system. We demonstrate that its partner protein SmpB binds the tRNA-like region (TLD) in vivo and chaperones the fold of the TLD-H2 region. We use an original approach combining the observation of tmRNA degradation pathways in a heterologous system, the analysis of the tmRNA digests by MS and NMR, and co-overproduction assays of tmRNA and SmpB. We study the conformation in solution of tmRNA alone or in complex with one SmpB before ribosome binding using SAXS. Our data show that Mg2+ drives compaction of the RNA structure and that, in the absence of Mg2+, SmpB has a similar effect albeit to a lesser extent. Our results show that tmRNA is intrinsically structured in solution with identical topology to that observed on complexes on ribosomes which should facilitate its subsequent recruitment by the 70S ribosome, free or preloaded with one SmpB molecule

Targeting the Proangiogenic VEGF-VEGFR Protein-Protein Interface with Drug-like Compounds by In Silico and In Vitro Screening

International audienceProtein-protein interactions play a central role in medicine, and their modulation with small organic compounds remains an enormous challenge. Because it has been noted that the macromolecular complexes modulated to date have a relatively pronounced binding cavity at the interface, we decided to perform screening experiments over the vascular endothelial growth factor receptor (VEGFR), a validated target for antiangiogenic treatments with a very flat interface. We focused the study on the VEGFR-1 D2 domain, and 20 active compounds were identified. These small compounds contained a (3-carboxy-2-ureido)thiophen unit and had IC(50) values in the low micromolar range. The most potent compound inhibited the VEGF-induced VEGFR-1 transduction pathways. Our findings suggest that our best hit may be a promising scaffold to probe this macromolecular complex and for the development of treatments of VEGFR-1-dependent diseases

Biophysical Studies of the Induced Dimerization of Human VEGF Receptor 1 Binding Domain by Divalent Metals Competing with VEGF-A.

Angiogenesis is tightly regulated through the binding of vascular endothelial growth factors (VEGFs) to their receptors (VEGFRs). In this context, we showed that human VEGFR1 domain 2 crystallizes in the presence of Zn2+, Co2+ or Cu2+ as a dimer that forms via metal-ion interactions and interlocked hydrophobic surfaces. SAXS, NMR and size exclusion chromatography analyses confirm the formation of this dimer in solution in the presence of Co2+, Cd2+ or Cu2+. Since the metal-induced dimerization masks the VEGFs binding surface, we investigated the ability of metal ions to displace the VEGF-A binding to hVEGFR1: using a competition assay, we evidenced that the metals displaced the VEGF-A binding to hVEGFR1 extracellular domain binding at micromolar level

Size-exclusion chromatography reveals the Cu²⁺ induced hVEGFR1d2 dimerization.

<p>(A) hVEGFR1d2 eluted either in the presence of either 1 mM EDTA (Purple curve) or 1 mM CuSO₄ (Green curve). The chromatogram revealed a single peak in the presence of 1 mM EDTA (d), and two major peaks corresponding to the monomer (b) and the dimer (a) in the presence of Cu²⁺. A third peak (c) consisting in Cu²⁺ ions eluted near the total volume. (B) SDS page of loaded samples and eluted peaks. hVEGFR1-d2 + CuCl₂ (Green): Loaded sample (ls), Ve = 11.3 ml (a), Ve = 14.1 ml (b); hVEGFR1-d2 + EDTA (Purple): Loaded sample (ls), Ve = 17.3 ml (c), Ve = 14.0 ml (d).</p