Valéry Larue

The structure of “defective in induced resistance” protein of Arabidopsis thaliana, DIR1, reveals a new type of lipid transfer protein

Screening of transfer DNA (tDNA) tagged lines of Arabidopsis thaliana for mutants defective in systemic acquired resistance led to the characterization of dir1‐1 (defective in induced resistance [systemic acquired resistance, SAR]) mutant. It has been suggested that the protein encoded by the dir1 gene, i.e., DIR1, is involved in the long distance signaling associated with SAR. DIR1 displays the cysteine signature of lipid transfer proteins, suggesting that the systemic signal could be lipid molecules. However, previous studies have shown that this signature is not sufficient to define a lipid transfer protein, i.e., a protein capable of binding lipids. In this context, the lipid binding properties and the structure of a DIR1–lipid complex were both determined by fluorescence and X‐ray diffraction. DIR1 is able to bind with high affinity two monoacylated phospholipids (dissociation constant in the nanomolar range), mainly lysophosphatidyl cholines, side‐by‐side in a large internal tunnel. Although DIR1 shares some structural and lipid binding properties with plant LTP2, it displays some specific features that define DIR1 as a new type of plant lipid transfer protein. The signaling function associated with DIR1 may be related to a specific lipid transport that needs to be characterized and to an additional mechanism of recognition by a putative receptor, as the structure displays on the surface the characteristic PxxP structural motif reminiscent of SH3 domain signaling pathways.

Structure-activity relationship analysis of the peptide deformylase inhibitor 5-bromo-1H-indole-3-acetohydroxamic acid.

SAR by NMR: A series of indole compounds derived from 5‐bromo‐1H‐indole‐3‐acetohydroxamic acid were synthesized. Their inhibitory activities were evaluated against purified peptide deformylases (PDFs), and their antibacterial activities against B. subtilis, E. coli (wild type and tolC), and a variety of pathogens were also determined. The potency of the best inhibitors was related to the NMR footprints of the respective acids with 15N‐labeled E. coli Ni‐PDF.

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

A 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.

Aminoglycoside and glycopeptide renal toxicity in intensive care patients studied by proton magnetic resonance spectroscopy of urine.

ObjectiveAminoglycoside and glycopeptide antibiotics are responsible for renal toxicity. In most cases, the nephrotoxicity is limited to a reversible tubular injury, but an acute and sustained renal failure may occur. The aim of our study was to explore the renal function of patients given these antimicrobial agents with proton magnetic resonance spectroscopy of urine. This technique is able to detect, in urine samples, a wide range of metabolites reflecting renal tubular function. The variables assessed by magnetic resonance spectroscopy were compared with the routine markers of renal function: creatinine, urea, and 24-hr urine volume. DesignProspective clinical study. SettingIntensive care unit. PatientsAll patients in an intensive care unit receiving an aminoglycoside and/or a glycopeptide were included in the study if they presented with signs of renal dysfunction. All experiments were performed on urine samples collected for the routine follow-up of these patients. InterventionProton spectra were acquired with water suppression, and the peak intensity of each metabolite was reported in relationship to the intensity of the creatinine peak. Measurements and Main ResultsThe ratio values obtained by magnetic resonance spectroscopy were compared with the values of creatininemia and blood urea obtained routinely by biochemistry and with the value of the 24-hr urine volume by logistic regression and general linear models. This statistical analysis showed that the ratio of dimethylamine to creatinine was highly correlated with creatininemia. ConclusionsDimethylamine is an osmolyte released from the medullar region of the kidney. Thus, our study demonstrated that nephrotoxicity from aminoglycosides and glycopeptides is not limited to proximal tubular toxicity but also may involve the medullar region (Henle loop and collecting duct) of the nephron.

Investigation of RNA–Ligand Interactions by 19F NMR Spectroscopy Using Fluorinated Probes†

Ribonucleic acid (RNA) is now recognized as playing a key role in many biological functions, and is emerging as an important new drug target. However, its therapeutic potential is still underexploited. Indeed, the limited understanding of the interactions between small molecules and RNA still hampers rational drug development of RNA-targeting molecules. Among the different methods available to investigate binding between small molecules and RNA, NMR spectroscopy is particularly attractive as it can deliver information on molecular interactions at the atomic level, including conformational rearrangements that can occur before or upon binding. The dynamic nature of this interaction is particularly important in RNA-regulated pathways. Many NMR spectroscopy techniques have been developed to visualize dynamic RNA–ligand interactions, most of them based on the observation of either the target or ligand H nuclei. However, some difficulties can occur when studying larger strands of RNA, as the number of detectable signals will increase. Introducing a specific label is one way to overcome this problem. An elegant method based on F NMR spectroscopy was proposed some years ago by Micura and co-workers. Introduction of a fluorine atom at a specific position of RNA allows local monitoring of binding events at this site. One technical difficulty with this approach is the need to chemically modify the RNA, which can be difficult for large RNAs or for RNAs with modified nucleotides. Furthermore, this modification can affect RNA– ligand interactions. We have recently shown that F NMR spectroscopy can be used to monitor the binding of racemic fluorinated molecules to various RNAs and that chiral recognition can be used to monitor the local conformation of the binding site. The use of ligand-based binding-competition NMR screening using fluorinated ligands has been described by Dalvit for the investigation of protein–ligand interactions (FAXS technique), but has not been applied to the study small molecules interacting with RNA. We report herein that competitive binding of fluorinated probes can be used to detect and quantify the interaction between unlabeled RNA and non-fluorinated ligands and to monitor dynamic RNA folding events (Figure 1).

Bacillus subtilis RNA deprotection enzyme RppH recognizes guanosine in the second position of its substrates

The initiation of mRNA degradation often requires deprotection of its 5′ end. In eukaryotes, the 5′-methylguanosine (cap) structure is principally removed by the Nudix family decapping enzyme Dcp2, yielding a 5′-monophosphorylated RNA that is a substrate for 5′ exoribonucleases. In bacteria, the 5′-triphosphate group of primary transcripts is also converted to a 5′ monophosphate by a Nudix protein called RNA pyrophosphohydrolase (RppH), allowing access to both endo- and 5′ exoribonucleases. Here we present the crystal structures of Bacillus subtilis RppH (BsRppH) bound to GTP and to a triphosphorylated dinucleotide RNA. In contrast to Bdellovibrio bacteriovorus RppH, which recognizes the first nucleotide of its RNA targets, the B. subtilis enzyme has a binding pocket that prefers guanosine residues in the second position of its substrates. The identification of sequence specificity for RppH in an internal position was a highly unexpected result. NMR chemical shift mapping in solution shows that at least three nucleotides are required for unambiguous binding of RNA. Biochemical assays of BsRppH on RNA substrates with single-base–mutation changes in the first four nucleotides confirm the importance of guanosine in position two for optimal enzyme activity. Our experiments highlight important structural and functional differences between BsRppH and the RNA deprotection enzymes of distantly related bacteria.

The PIR domain of Grb14 is an intrinsically unstructured protein: implication in insulin signaling.

Grb14 belongs to the Grb7 family of adapter proteins and was identified as a negative regulator of insulin signal transduction. Its inhibitory effect on the insulin receptor kinase activity is controlled by a newly discovered domain called PIR. To investigate the biochemical and biophysical characteristics of this new domain, we cloned and purified recombinant PIR‐SH2, PIR, and SH2 domains. The isolated PIR and PIR‐SH2 domains were physiologically active and inhibited insulin‐induced reinitiation of meiosis in the Xenopus oocytes system. However, NMR experiments on 15N‐labelled PIR revealed that it did not present secondary structure. These results suggest that the PIR domain belongs to the growing family of intrinsically unstructured proteins.

Magnetic resonance spectroscopy of cellular lipid extracts from sensitive, resistant and reverting K562 cells and flow cytometry for investigating the P-glycoprotein function in resistance reversion

The proton NMR spectra of K562 cells contain resonances of lipids. When these cells acquire multidrug resistance phenotype, the NMR lipid signals are modified and partially recovered when the resistance is reversed. The goals of the present study are to elucidate the mechanism of the resistance phenotype reversion and to investigate the possible origin of lipid signals detected in whole cells with proton NMR spectroscopy. Therefore, the K562 drug‐sensitive cell line, its adriamycin resistant counterpart and two reverting derivates, obtained by verapamil treatment and long term culture in drug‐free medium, were used in this study. The P‐glycoprotein (P‐gp) pump function was measured by flow cytometry and lipids were extracted to be analysed by proton and phosphorus spectroscopy. The phenotype reversion is due to the decrease of the P‐gp function and an increased entrance of anthracycline drug when compared with the resistant cells. The spectra obtained on extracts showed no modification of the fatty acid composition and of the ratio of total cholesterol to fatty acid content. A different phospholipid composition in sensitive and resistant cells was found, but the reversion of resistance did not produce a recovery of these lipids. Thus, the lipid NMR spectra of extracts could not explain the spectral modifications observed on whole cells, in relation to acquiring and reverting drug resistance. These results are in favour of a different lipid organization or of localization within the cell. Copyright

Fragment-based design of small RNA binders: promising developments and contribution of NMR.

Fragment-based drug design has become increasingly popular over the last decade. We review here the use of this approach to design small RNA binders. In addition, we discuss the use of NMR to detect the binding of small molecules on RNA targets and to guide chemists in the design of compounds targeting RNA.

The crystal structure of oxylipin-conjugated barley LTP1 highlights the unique plasticity of the hydrophobic cavity of these plant lipid-binding proteins.

The barley lipid transfer protein (LTP1) adducted by an alpha-ketol, (9-hydroxy-10-oxo-12(Z)-octadecenoic acid) exhibits an unexpected high lipid transfer activity. The crystal structure of this oxylipin-adducted LTP1, (LTP1b) was determined at 1.8A resolution. The covalently bound oxylipin was partly exposed at the surface of the protein and partly buried within the hydrophobic cavity. The structure of the oxylipidated LTP1 emphasizes the unique plasticity of the hydrophobic cavity of these plant lipid-binding proteins when compared to the other members of the family. The plasticity of the hydrophobic cavity and increase of its surface hydrophobicity induced by the oxylipin account for the improvement of the lipid transfer activity of LTP1b. These observations open new perspectives to explore the different biological functions of LTPs, including their allergenic properties.