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The Hepatitis C virus nonstructural protein 5A (NS5A) is a membrane-associated protein involved in multiple steps of the viral life cycle. Direct-acting antivirals (DAAs) targeting NS5A are a cornerstone of antiviral therapy, but the mode-of-action of these drugs is poorly understood. This is due to the lack of information on the membrane-bound NS5A structure. Herein, we present the structural model of an NS5A AH-linker-D1 protein reconstituted as proteoliposomes. We use highly sensitive proton-detected solid-state NMR methods suitable to study samples generated through synthetic biology approaches. Spectra analyses disclose that both the AH membrane anchor and the linker are highly flexible. Paramagnetic relaxation enhancements (PRE) reveal that the dimer organization in lipids requires a new type of NS5A self-interaction not reflected in previous crystal structures. In conclusion, we provide the first characterization of NS5A AH-linker-D1 in a lipidic environment shedding light onto the mode-of-action of clinically used NS5A inhibitors. ISSN:1433-7851 ISSN:1521-3773 ISSN:0570-0833

International audience; We sequentially assigned the fully-protonated capsids made from core proteins of the Hepatitis B virus using proton detection at 100 kHz magic-angle spinning (MAS) in 0.7 mm rotors and compare sensitivity and assignment completeness to previously obtained assignments using carbon-detection techniques in 3.2 mm rotors and 17.5 kHz MAS. We show that proton detection shows a global gain of a factor ∼50 in mass sensitivity, but that signal-to-noise ratios and completeness of the assignment was somewhat higher for carbon-detected experiments for comparable experimental times. We also show that deuteration and H N back protonation improves the proton linewidth at 100 kHz MAS by a factor of 1.5, from an average of 170-110 Hz, and by a factor of 1.3 compared to deuterated capsids at 60 kHz MAS in a 1.3 mm rotor. Yet, several H N protons cannot be back-exchanged due to solvent inaccessibility, which results in a total of 15% of the amides missing in the spectra.

International audience; Modulation of capsid assembly by small molecules has become a central concept in the fight against viral infection. Proper capsid assembly is crucial to form the high molecular weight structures that protect the viral genome and that, often in concert with the envelope, allow for cell entry and fusion. Atomic details underlying assembly modulation are generally studied using preassembled protein complexes, while the activity of assembly modulators during assembly remains largely open and poorly understood, as necessary tools are lacking. We here use the full-length hepatitis B virus (HBV) capsid protein (Cp183) as a model to present a combination of cell-free protein synthesis and solid-state NMR as an approach which shall open the possibility to produce and analyze the formation of higher-order complexes directly on exit from the ribosome. We demonstrate that assembled capsids can be synthesized in amounts sufficient for structural studies, and show that addition of assembly modulators to the cell-free reaction produces objects similar to those obtained by addition of the compounds to preformed Cp183 capsids. These results establish the cell-free system as a tool for the study of capsid assembly modulation directly after synthesis by the ribosome, and they open the perspective of assessing the impact of natural or synthetic compounds, or even enzymes that perform post-translational modifications, on capsids structures.

Spectral resolution is the key to unleashing the structural and dynamic information contained in NMR spectra. Fast magic‐angle spinning (MAS) has recently revolutionized the spectroscopy of biomolecular solids. Herein, we report a further remarkable improvement in the resolution of the spectra of four fully protonated proteins and a small drug molecule by pushing the MAS rotation frequency higher (150 kHz) than the more routinely used 100 kHz. We observed a reduction in the average homogeneous linewidth by a factor of 1.5 and a decrease in the observed linewidth by a factor 1.25. We conclude that even faster MAS is highly attractive and increases mass sensitivity at a moderate price in overall sensitivity., ChemBioChem, 21 (17), ISSN:1439-4227, ISSN:1439-7633

We sequentially assigned the fully-protonated capsids made from core proteins of the Hepatitis B virus using proton detection at 100 kHz magic-angle spinning (MAS) in 0.7 mm rotors and compare sensitivity and assignment completeness to previously obtained assignments using carbon-detection techniques in 3.2 mm rotors and 17.5 kHz MAS. We show that proton detection shows a global gain of a factor ~50 in mass sensitivity, but that signal-to-noise ratios and completeness of the assignment was somewhat higher for carbon-detected experiments for comparable experimental times. We also show that deuteration and HN back protonation improves the proton linewidth at 100 kHz MAS by a factor of 1.5, from an average of 170–110 Hz, and by a factor of 1.3 compared to deuterated capsids at 60 kHz MAS in a 1.3 mm rotor. Yet, several HN protons cannot be back-exchanged due to solvent inaccessibility, which results in a total of 15% of the amides missing in the spectra., Frontiers in Molecular Biosciences, 6, ISSN:2296-889X

Aiming at the design of an allosteric modulator of the neonatal Fc receptor (FcRn)–Immunoglobulin G (IgG) interaction, we developed a new methodology including NMR fragment screening, X-ray crystallography, and magic-angle-spinning (MAS) NMR at 100 kHz after sedimentation, exploiting very fast spinning of the nondeuterated soluble 42 kDa receptor construct to obtain resolved proton-detected 2D and 3D NMR spectra. FcRn plays a crucial role in regulation of IgG and serum albumin catabolism. It is a clinically validated drug target for the treatment of autoimmune diseases caused by pathogenic antibodies via the inhibition of its interaction with IgG. We herein present the discovery of a small molecule that binds into a conserved cavity of the heterodimeric, extracellular domain composed of an α-chain and β2-microglobulin (β2m) (FcRnECD, 373 residues). X-ray crystallography was used alongside NMR at 100 kHz MAS with sedimented soluble protein to explore possibilities for refining the compound as an allosteric modulator. Proton-detected MAS NMR experiments on fully protonated [13C,15N]-labeled FcRnECD yielded ligand-induced chemical-shift perturbations (CSPs) for residues in the binding pocket and allosteric changes close to the interface of the two receptor heterodimers present in the asymmetric unit as well as potentially in the albumin interaction site. X-ray structures with and without ligand suggest the need for an optimized ligand to displace the α-chain with respect to β2m, both of which participate in the FcRnECD–IgG interaction site. Our investigation establishes a method to characterize structurally small molecule binding to nondeuterated large proteins by NMR, even in their glycosylated form, which may prove highly valuable for structure-based drug discovery campaigns., PLoS Biology, 16 (5), ISSN:1544-9173, ISSN:1545-7885

ChemBioChem, 21 (10), ISSN:1439-4227, ISSN:1439-7633

The Hepatitis C virus nonstructural protein 5A (NS5A) is a membrane-associated protein involved in multiple steps of the viral life cycle. Direct-acting antivirals (DAAs) targeting NS5A are a cornerstone of antiviral therapy, but the mode-of-action of these drugs is poorly understood. This is due to the lack of information on the membrane-bound NS5A structure. Herein, we present the structural model of an NS5A AH-linker-D1 protein reconstituted as proteoliposomes. We use highly sensitive proton-detected solid-state NMR methods suitable to study samples generated through synthetic biology approaches. Spectra analyses disclose that both the AH membrane anchor and the linker are highly flexible. Paramagnetic relaxation enhancements (PRE) reveal that the dimer organization in lipids requires a new type of NS5A self-interaction not reflected in previous crystal structures. In conclusion, we provide the first characterization of NS5A AH-linker-D1 in a lipidic environment shedding light onto the mode-of-action of clinically used NS5A inhibitors., Angewandte Chemie. International Edition, 60 (10), ISSN:1433-7851, ISSN:1521-3773, ISSN:0570-0833

Journal of Biomolecular NMR, 65 (2), ISSN:0925-2738, ISSN:1573-5001

Modulation of capsid assembly by small molecules has become a central concept in the fight against viral infection. Proper capsid assembly is crucial to form the high molecular weight structures that protect the viral genome and that, often in concert with the envelope, allow for cell entry and fusion. Atomic details underlying assembly modulation are generally studied using preassembled protein complexes, while the activity of assembly modulators during assembly remains largely open and poorly understood, as necessary tools are lacking. We here use the full-length hepatitis B virus (HBV) capsid protein (Cp183) as a model to present a combination of cell-free protein synthesis and solid-state NMR as an approach which shall open the possibility to produce and analyze the formation of higher-order complexes directly on exit from the ribosome. We demonstrate that assembled capsids can be synthesized in amounts sufficient for structural studies, and show that addition of assembly modulators to the cell-free reaction produces objects similar to those obtained by addition of the compounds to preformed Cp183 capsids. These results establish the cell-free system as a tool for the study of capsid assembly modulation directly after synthesis by the ribosome, and they open the perspective of assessing the impact of natural or synthetic compounds, or even enzymes that perform post-translational modifications, on capsids structures., Frontiers in Molecular Biosciences, 6, ISSN:2296-889X