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In NMR spectroscopy, residual dipolar couplings (RDCs) have emerged as one of the most exquisite probes of biological structure and dynamics. The measurement of RDCs relies on the partial alignment of the molecule of interest, for example by using a liquid crystal as a solvent. Here, we establish bacterial type 1 pili as an alternative liquid-crystalline alignment medium for the measurement of RDCs. To achieve alignment at pilus concentrations that allow for efficient NMR sample preparation, we elongated wild-type pili by recombinant overproduction of the main structural pilus subunit. Building on the extraordinary stability of type 1 pili against spontaneous dissociation and unfolding, we show that the medium is compatible with challenging experimental conditions such as high temperature, the presence of detergents, organic solvents or very acidic pH, setting it apart from most established alignment media. Using human ubiquitin, HIV-1 TAR RNA and camphor as spectroscopic probes, we demonstrate the applicability of the medium for the determination of RDCs of proteins, nucleic acids and small molecules. Our results show that type 1 pili represent a very useful alternative to existing alignment media and may readily assist the characterization of molecular structure and dynamics by NMR., Angewandte Chemie. International Edition, ISSN:1433-7851, ISSN:1521-3773, ISSN:0570-0833

Objective. The aim of this work was to highlight and characterize a systemic ‘star-like’ artefact inherent to the low field 0.35 T MRIdian MR-linac system, a magnetic resonance guided radiotherapy device. This artefact is induced by the original split gradients coils design. This design causes a surjection of the intensity gradient in Z (or head-feet) direction. This artefact appears on every sequence with phase encoding in the head-feet direction. Approach. Basic gradient echo sequence and clinical mandatory bSSFP sequence were used. Three setups using manufacturer provided QA phantoms were designed: two including the linearity control grid used for the characterisation and a third including two homogeneity control spheres dedicated to the artefact management in a more clinical like situation. The presence of the artefact was checked in four different MRidian sites. The tested parameters based on the literature were: phase encoding orientation, slab selectivity, excitation bandwidth (BWRF), acceleration factor (R) and phase/slab oversampling (PO/SO). Main results. The position of this artefact is constant and reproducible over the tested MRIdian sites. The typical singularity saturated dot or star is visible even with the 3D slab-selection enabled. A management is proposed by decreasing the BWRF, the R in head-feet direction and increasing the PO/SO. The oversampling can be optimized using a formula to anticipate the location of artefact in the field of view. Significance. The star-like artefact has been well characterised. A manageable solution comes at the cost of acquisition time. Observed in clinical cases, the artefact may degrade the images used for the RT planning and repositioning during the treatment unless corrected.

Intensity modulation treatments are widely used in radiotherapy because of many known advantages. In this context, the picket fence test (PF) is a relevant test to check the Multileaf Collimator (MLC) performances. So this work compares and evaluates three analysis platforms for the PF used routinely by three different institutions. This study covers two linear accelerators (Linac) with two MLC types, a Millenium 120 MLC and Millenium 120 High Definition MLC respectively on a Varian Truebeam and Truebeam STx. Both linacs include an As 1200 portal imager (EPID). From a reference PF plan, MLC errors have been introduced to modify the slits in position or width (shifts from 0.1 to 0.5 mm on one or both banks). Then errors have been defined on the EPID to investigate detection system deviations (signal sensitivity and position variations). Finally, 110 DICOM-RT images have been generated and analyzed by each software system. All software systems have shown good performances to quantify the position errors, even though the leaf pair identifications can be wrong in some cases regarding the analysis method considered. The slit width measurement (not calculated by all software systems) has shown good sensitivity, but some quantification difficulties have been highlighted regardless of the analysis method used. Linked to the expected accuracy of the PF test, the imager variations have demonstrated considerable influence in the results. Differences in the results and the analysis methods have been pointed out for each software system. The results can be helpful to optimize the settings of each analysis software system depending on expectations and treatment modalities of each institution.

The journal of physical chemistry / B 124(49), 11089-11097 (2020). doi:10.1021/acs.jpcb.0c08150 special issue: "Hellmut Eckert Festschrift : virtual issue", Published by American Chemical Society, Washington, DC

The molecular mechanism that initiates the synthesis of starch granules is poorly understood. Here, we discovered two plastidial proteins involved in granule initiation in Arabidopsis thaliana leaves. Both contain coiled coils and a family-48 carbohydrate binding module (CBM48) and are homologs of the PROTEIN TARGETING TO STARCH (PTST) protein; thus, we named them PTST2 and PTST3. Chloroplasts in mesophyll cells typically contain five to seven granules, but remarkably, most chloroplasts in ptst2 mutants contained zero or one large granule. Chloroplasts in ptst3 had a slight reduction in granule number compared with the wild type, while those of the ptst2 ptst3 double mutant contained even fewer granules than ptst2. The ptst2 granules were larger but similar in morphology to wild-type granules, but those of the double mutant had an aberrant morphology. Immunoprecipitation showed that PTST2 interacts with STARCH SYNTHASE4 (SS4), which influences granule initiation and morphology. Overexpression of PTST2 resulted in chloroplasts containing many small granules, an effect that was dependent on the presence of SS4. Furthermore, isothermal titration calorimetry revealed that the CBM48 domain of PTST2, which is essential for its function, interacts with long maltooligosaccharides. We propose that PTST2 and PTST3 are critical during granule initiation, as they bind and deliver suitable maltooligosaccharide primers to SS4.

Human antigen R (HuR) is a key regulator of cellular mRNAs containing adenylate/uridylate–rich elements (AU-rich elements; AREs). These are a major class of cis elements within 3′ untranslated regions, targeting these mRNAs for rapid degradation. HuR contains three RNA recognition motifs (RRMs): a tandem RRM1 and 2, followed by a flexible linker and a C-terminal RRM3. While RRM1 and 2 are structurally characterized, little is known about RRM3. Here we present a 1.9-Å-resolution crystal structure of RRM3 bound to different ARE motifs. This structure together with biophysical methods and cell-culture assays revealed the mechanism of RRM3 ARE recognition and dimerization. While multiple RNA motifs can be bound, recognition of the canonical AUUUA pentameric motif is possible by binding to two registers. Additionally, RRM3 forms homodimers to increase its RNA binding affinity. Finally, although HuR stabilizes ARE-containing RNAs, we found that RRM3 counteracts this effect, as shown in a cell-based ARE reporter assay and by qPCR with native HuR mRNA targets containing multiple AUUUA motifs, possibly by competing with RRM12., Proceedings of the National Academy of Sciences of the United States of America, 116 (8), ISSN:0027-8424, ISSN:1091-6490

DNA replication is a crucial stage in the transfer of genetic information from parent to daughter cells. This mechanism involves multiple proteins with one key player being the primase. Primases are single-stranded DNA dependent RNA polymerases. On the leading strand, they synthesize the primer once allowing DNA elongation while on the lagging strand primers are generated repeatedly (Okazaki fragments). Primases have the unique ability to create the first phosphodiester bond yielding a dinucleotide which is initially elongated by primases and then by DNA polymerases. Primase activity has been studied in the last decades but the detailed molecular steps explaining some unique features remain unclear. High-resolution structures of free and bound primases domains have brought significant insights in the understanding of the primase reaction cycle. Here, we give a short review of the structural work conducted in the field of archaeo-eukaryotic primases and we underline the missing “pictures” of the active forms of the enzyme which are of major interest. We organized our analysis with respect to the progression through the catalytic pathway., Computational and Structural Biotechnology Journal, 13, ISSN:2001-0370

N6A methylation is the most abundant RNA modification occurring within messenger RNA. Impairment of methylase or demethylase functions are associated with severe phenotypes and diseases in several organisms. Beside writer and eraser enzymes of this dynamic RNA epigenetic modification, reader proteins that recognize this modification are involved in numerous cellular processes. Although the precise characterization of these reader proteins remains unknown, preliminary data showed that most potential reader proteins contained a conserved YT521-B homology (YTH) domain. Here we define the YTH domain of rat YT521-B as a N6-methylated adenosine reader domain and report its solution structure in complex with a N6-methylated RNA. The structure reveals a binding preference for NGANNN RNA hexamer and a deep hydrophobic cleft for m6A recognition. These findings establish a molecular function for YTH domains as m6A reader domains and should guide further studies into the biological functions of YTH-containing proteins in m6A recognition., Nucleic Acids Research, 42 (22), ISSN:1362-4962, ISSN:0301-5610

Bioinformatics, 31 (18), ISSN:1367-4803, ISSN:1460-2059

Ribonucleoproteins (RNPs) are key regulators of cellular function. We established an efficient approach, crosslinking of segmentally isotope-labeled RNA and tandem mass spectrometry (CLIR-MS/MS), to localize protein-RNA interactions simultaneously at amino acid and nucleotide resolution. The approach was tested on polypyrimidine tract binding protein 1 and U1 small nuclear RNP. Our method provides distance restraints to support integrative atomic-scale structural modeling and to gain mechanistic insights into RNP-regulated processes.

International audience; A new nonlinear and variational homogenization procedure that incorporates the fluctuations of the plastic strains within the phases is proposed to assess the local and effective responses of elasto-(visco)plastic composites with local work-hardening. The formulation is an extension of the EIV approach of Lahellec and Suquet (2007a) initially proposed for elasto-viscoplastic composites without both threshold and hardening. This extension relies both on an appropriate linearization of the stored-energy function which accounts for the isotropic and kinematic hardening and an incorporation of the elastic unloading. The accuracy of the approach is then assessed by comparisons with reference numerical solutions and other nonlinear homogenization schemes of the literature carried out on both porous elasto-plastic materials and several reinforced elasto-(visco)plastic composites subjected to cyclic proportional loadings.

In absence of beta-lactam antibiotics, BlaI and MecI homodimeric repressors negatively control the expression of genes involved in beta-lactam resistance in Bacillus licheniformis and in Staphylococcus aureus. Subsequently to beta-lactam presence, BlaI/MecI is inactivated by a single-point proteolysis that separates its N-terminal DNA-binding domain to its C-terminal domain responsible for its dimerization. Concomitantly to this proteolysis, the truncated repressor acquires a low affinity for its DNA target that explains the expression of the structural gene for resistance. To understand the loss of the high DNA affinity of the truncated repressor, we have determined the different dissociation constants of the system and solved the solution structure of the B. licheniformis monomeric repressor complexed to the semi-operating sequence OP1 of blaP (1/2OP1blaP) by using a de novo docking approach based on inter-molecular nuclear Overhauser effects and chemical-shift differences measured on each macromolecular partner. Although the N-terminal domain of the repressor is not subject to internal structural rearrangements upon DNA binding, the molecules adopt a tertiary conformation different from the crystallographic operator-repressor dimer complex, leading to a 30 degrees rotation of the monomer with respect to a central axis extended across the DNA. These results open new insights for the repression and induction mechanisms of bacterial resistance to beta-lactams.

The trimeric influenza virus polymerase, comprising subunits PA, PB1 and PB2, is responsible for transcription and replication of the segmented viral RNA genome. Using a novel library-based screening technique called expression of soluble proteins by random incremental truncation (ESPRIT), we identified an independently folded C-terminal domain from PB2 and determined its solution structure by NMR. Using green fluorescent protein fusions, we show that both the domain and the full-length PB2 subunit are efficiently imported into the nucleus dependent on a previously overlooked bipartite nuclear localization sequence (NLS). The crystal structure of the domain complexed with human importin alpha5 shows how the last 20 residues unfold to permit binding to the import factor. The domain contains three surface residues implicated in adaptation from avian to mammalian hosts. One of these tethers the NLS-containing peptide to the core of the domain in the unbound state.

Proteins that bind single-stranded nucleic acids have crucial roles in cells, and structural analyses have contributed to a better understanding of their functions. In this issue of Structure, Dickey and colleagues describe several high resolution structures of a single OB-fold bound to different single-stranded DNA (ssDNA) sequences and reveal a spectacular co-adaptability of the protein/ssDNA interactions.

Journal of Biomolecular NMR, 56 (2), ISSN:0925-2738, ISSN:1573-5001

The ampG gene codes for a permease required to uptake anhydro-muropeptides into bacterial cytoplasm. Located upstream in the same operon, is another 579-base-pair-long open reading frame encoding a putative lipoprotein YajG, whose nearly complete 1H,13C,15N assignments are reported here.

The hepatitis C virus (HCV) is a positive-strand RNA virus belonging to the Flaviviridae . Its genome carries at either end highly conserved nontranslated regions (NTRs) containing cis -acting RNA elements that are crucial for replication. In this study, we identified a novel RNA element within the NS5B coding sequence that is indispensable for replication. By using secondary structure prediction and nuclear magnetic resonance spectroscopy, we found that this RNA element, designated 5BSL3.2 by analogy to a recent report (S. You, D. D. Stump, A. D. Branch, and C. M. Rice, J. Virol. 78: 1352-1366, 2004), consists of an 8-bp lower and a 6-bp upper stem, an 8-nucleotide-long bulge, and a 12-nucleotide-long upper loop. Mutational disruption of 5BSL3.2 structure blocked RNA replication, which could be restored when an intact copy of this RNA element was inserted into the 3′ NTR. By using this replicon design, we mapped the elements in 5BSL3.2 that are critical for RNA replication. Most importantly, we discovered a nucleotide sequence complementarity between the upper loop of this RNA element and the loop region of stem-loop 2 in the 3′ NTR. Mismatches introduced into the loops inhibited RNA replication, which could be rescued when complementarity was restored. These data provide strong evidence for a pseudoknot structure at the 3′ end of the HCV genome that is essential for replication.

To resist to β-lactam antibiotics Eubacteria either constitutively synthesize a β-lactamase or a low affinity penicillin-binding protein target, or induce its synthesis in response to the presence of antibiotic outside the cell. In Bacillus licheniformis and Staphylococcus aureus, a membrane-bound penicillin receptor (BlaR/MecR) detects the presence of β-lactam and launches a cytoplasmic signal leading to the inactivation of BlaI/MecI repressor, and the synthesis of a β-lactamase or a low affinity target. We identified a dipeptide, resulting from the peptidoglycan turnover and present in bacterial cytoplasm, which is able to directly bind to the BlaI/MecI repressor and to destabilize the BlaI/MecI-DNA complex. We propose a general model, in which the acylation of BlaR/MecR receptor and the cellular stress induced by the antibiotic, are both necessary to generate a cell wall-derived coactivator responsible for the expression of an inducible β-lactam-resistance factor. The new model proposed confirms and emphasizes the role of peptidoglycan degradation fragments in bacterial cell regulation., Author Summary Beta-lactamases are enzymes produced by some bacteria and are responsible for their resistance to beta-lactam antibiotics like penicillin. Among these bacteria some of them possess a beta-lactamase that is only produced at a high level when a beta-lactam antibiotic is present outside the cell. This mechanism of regulation, named beta-lactamase induction, has been described both in Gram-negative and Gram-positive bacteria. In Staphylococcus aureus and Bacillus licheniformis, two Gram-positive bacteria, their beta-lactamase is induced by homologous proteins including a membrane-bound penicillin receptor, BlaR1, and a cytoplasmic DNA-binding protein, BlaI, acting as repressor. The first step in the induction mechanism is the acylation of the extracytoplasmic domain of BlaR receptor by the beta-lactam antibiotic concomitant with the activation of the peptidase activity of its cytoplasmic domain. The activated receptor launched a cytoplasmic signal leading to the inactivation of BlaI repressor. The nature of this cytoplasmic signal is not well understood. In our study, we identified that a dipeptide issued from the bacterial cell wall is able to inactivate the BlaI repressor. It is the first time that a cell wall fragment is shown to reenter in the cytoplasm of Gram-positive bacteria to regulate gene expression.