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Combination of solution and solid state NMR yields a molecular level view of the interactions between antibiotic teixobactin and bacterial cell wall building block lipid II., Teixobactin is a new promising antibiotic that targets cell wall biosynthesis by binding to lipid II and has no detectable resistance thanks to its unique but yet not fully understood mechanism of operation. To aid in the structure-based design of teixobactin analogues with improved pharmacological properties, we present a 3D structure of native teixobactin in membrane mimetics and characterise its binding to lipid II through a combination of solution NMR and fast (90 kHz) magic angle spinning solid state NMR. In NMR titrations, we observe a pattern strongly suggesting interactions between the backbone of the C-terminal “cage” and the pyrophosphate moiety in lipid II. We find that the N-terminal part of teixobactin does not only act as a membrane anchor, as previously thought, but is actively involved in binding. Moreover, teixobactin forms a well-structured and specific complex with lipid II, where the N-terminal part of teixobactin assumes a β conformation that is highly prone to aggregation, which likely contributes to the antibiotic's high bactericidal efficiency. Overall, our study provides several new clues to teixobactin's modes of action.

In bacteria, nucleoid associated proteins (NAPs) take part in active chromosome organization by supercoil management, three-dimensional DNA looping and direct transcriptional control. Mycobacterial integration host factor (mIHF, rv1388) is a NAP restricted to Actinobacteria and essential for survival of the human pathogen Mycobacterium tuberculosis. We show in vitro that DNA binding by mIHF strongly stabilizes the protein and increases its melting temperature. The structure obtained by Nuclear Magnetic Resonance (NMR) spectroscopy characterizes mIHF as a globular protein with a protruding alpha helix and a disordered N-terminus, similar to Streptomyces coelicolor IHF (sIHF). NMR revealed no residues of high flexibility, suggesting that mIHF is a rigid protein overall that does not undergo structural rearrangements. We show that mIHF only binds to double stranded DNA in solution, through two DNA binding sites (DBSs) similar to those identified in the x-ray structure of sIHF. According to Atomic Force Microscopy, mIHF is able to introduce left-handed loops of ca. 100 nm size (∼300 bp) in supercoiled cosmids, thereby unwinding and relaxing the DNA.

Journal of Biomolecular NMR, 42 (1), ISSN:0925-2738, ISSN:1573-5001

http://thcgerm.free.fr/spip.php?article123; National audience; Structural biology studies provide key insights for life sciences, ranging from drug discovery to biofuel engineering, and from understanding metabolic disease mechanisms to improve human and animal health. Nuclear Magnetic Resonance (NMR) spectroscopy enables high-resolution three-dimensional studies of biomolecules and provides unique information on molecular dynamics and interactions. The downside of protein structure determination by NMR is however the elaborated data analysis workflow, involving many imperfect experimental and computational steps. In fact, NMR-only driven structure determination projects will always be limited by the intrinsic experimental imperfection of the technique, therefore truly robust, objective and highly accurate data analysis of large protein complexes (> 30kDa) is hard to achieve using NMR data only.The current limitations of NMR studies can however be bridged by simultaneous consideration of multiple types of information. A promising strategy consists in supplementing the conventional structure determination process with a prior knowledge extracted from databases. Here we will describe such an integrated, hybrid approach for NMR structure elucidation. We will present application examples for rapid and accurate structure determination of small to medium-sized soluble proteins and the de novo structure of a 28kDa protein dimer in a 2.5MDa viral capsid assembly by solid-state NMR.

International audience; The atomic-level characterization of large viral particles is one of the greatest challenges of modern structural biology, as well as a fundamental step for the design of effective antiviral treatments. Over the last decades, solid-state NMR (ssNMR) has developed into a powerful structural tool for studying structure and dynamics of solid biological samples at atomic resolution. However, the inherently low sensitivity and poor resolution of the technique has limited its applicability to small proteins that can be tightly packed at a high molar concentration, while large proteins or multi-domain assemblies were mostly inaccessible to site-specific ssNMR studies. This has been recently overcome by the introduction of faster spinning probes, which facilitate the use of proton-detected ssNMR experiments, as well as by dynamic nuclear polarization (DNP), which allows transfer of polarization from the unpaired electrons of a paramagnetic center to the surrounding nuclei, and can enhance the sensitivity of ssNMR experiments by several orders of magnitude. Here we demonstrate the effectiveness of the recently developed ssNMR methods employing proton detection at high field and 100 kHz MAS by structure determination of the 2.5 MDa icosahedral capsid of the AP205 bacteriophage. We show that at this spinning regime spectral resolution is high enough to detect resolved correlations from amide and side-chain protons of all residue types, and to reliably measure a dense network of 1H-1H proximities that define the dimeric capsid subunit structure. The subunit structure is then used in conjunction with a low resolution EM map to construct an atomic-level description of the global capsid architecture.Additionally, we show that high quality DNP NMR spectra of the AP205 nucleocapsid can be obtained by combining high magnetic field (800 MHz) and fast magic-angle spinning (40 kHz) .This enables assignment of aromatic resonances of the encapsidated RNA and the nucleoprotein, which are not observed at room temperature, opening up new possibilities for intermolecular interaction studies.

International audience; The WeNMR (http://www.wenmr.eu) project is an EU-fundedinternational effort to streamline and automate structuredetermination from Nuclear Magnetic Resonance (NMR) data.Conventionally calculation of structure requires the use of varioussoftwares, considerable user expertise and ample computationalresources. To facilitate the use of NMR spectroscopy in life sciencesthe eNMR/WeNMR consortium has set out to provide protocolizedservices through easy-to-use web interfaces, while still retainingsufficient flexibility to handle more specific requests. Thus far, anumber of programs often used in Structural Biology have beenmade available through portals, including HADDOCK, XPLOR-NIH,CYANA and CS-ROSETTA, MARS, MDDNMR. The implementationof these services, in particular the distribution of calculations to theGrid, involves a novel mechanism for submission and handling ofjobs that is independent of the type of job being run. With over 280registered users (April 2011), eNMR/WeNMR is currently one of thelargest Virtual Organization (VO) in life sciences. With its large andworldwide user community, WeNMR has become the first VirtualResearch

International audience

International audience

International audience

International audience