Fast Magic-Angle-Spinning NMR Reveals the Evasive Hepatitis†B Virus Capsid C-Terminal Domain.

Experimentally determined protein structures often feature missing domains. One example is the C-terminal domain (CTD) of the hepatitis B virus capsid protein, a functionally central part of this assembly, crucial in regulating nucleic-acid interactions, cellular trafficking, nuclear import, particle assembly and maturation. However, its structure remained elusive to all current techniques, including NMR. Here we show that the recently developed proton-detected fast magic-angle-spinning solid-state NMR at >100 kHz MAS allows one to detect this domain and unveil its structural and dynamic behavior. We describe the experimental framework used and compare the domain's behavior in different capsid states. The developed approaches extend solid-state NMR observations to residues characterized by large-amplitude motion on the microsecond timescale, and shall allow one to shed light on other flexible protein domains still lacking their structural and dynamic characterization.

A pocket-factor-triggered conformational switch in the hepatitis B virus capsid.

Viral hepatitis is growing into an epidemic illness, and it is urgent to neutralize the main culprit, hepatitis B virus (HBV), a small-enveloped retrotranscribing DNA virus. An intriguing observation in HB virion morphogenesis is that capsids with immature genomes are rarely enveloped and secreted. This prompted, in 1982, the postulate that a regulated conformation switch in the capsid triggers envelopment. Using solid-state NMR, we identified a stable alternative conformation of the capsid. The structural variations focus on the hydrophobic pocket of the core protein, a hot spot in capsid-envelope interactions. This structural switch is triggered by specific, high-affinity binding of a pocket factor. The conformational change induced by the binding is reminiscent of a maturation signal. This leads us to formulate the "synergistic double interaction" hypothesis, which explains the regulation of capsid envelopment and indicates a concept for therapeutic interference with HBV envelopment.

Protein NMR Spectroscopy at 150†kHz Magic-Angle Spinning Continues To Improve Resolution and Mass Sensitivity.

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.

Nucleotide Binding Modes in a Motor Protein Revealed by 31 P- and 1 H-Detected MAS Solid-State NMR Spectroscopy.

Protein-nucleic acid interactions play important roles not only in energy-providing reactions, such as ATP hydrolysis, but also in reading, extending, packaging, or repairing genomes. Although they can often be analyzed in detail with X-ray crystallography, complementary methods are needed to visualize them in complexes, which are not crystalline. Here, we show how solid-state NMR spectroscopy can detect and classify protein-nucleic interactions through site-specific 1 H- and 31 P-detected spectroscopic methods. The sensitivity of 1 H chemical-shift values on noncovalent interactions involved in these molecular recognition processes is exploited allowing us to probe directly the chemical bonding state, an information, which is not directly accessible from an X-ray structure. We show that these methods can characterize interactions in easy-to-prepare sediments of the 708 kDa dodecameric DnaB helicase in complex with ADP:AlF4- :DNA, and this despite the very challenging size of the complex.

Including Protons in Solid-State NMR Resonance Assignment and Secondary Structure Analysis: The Example of RNA Polymerase II Subunits Rpo4/7.

1H-detected solid-state NMR experiments feasible at fast magic-angle spinning (MAS) frequencies allow accessing 1H chemical shifts of proteins in solids, which enables their interpretation in terms of secondary structure. Here we present 1H and 13C-detected NMR spectra of the RNA polymerase subunit Rpo7 in complex with unlabeled Rpo4 and use the 13C, 15N, and 1H chemical-shift values deduced from them to study the secondary structure of the protein in comparison to a known crystal structure. We applied the automated resonance assignment approach FLYA including 1H-detected solid-state NMR spectra and show its success in comparison to manual spectral assignment. Our results show that reasonably reliable secondary-structure information can be obtained from 1H secondary chemical shifts (SCS) alone by using the sum of 1Hα and 1HN SCS rather than by TALOS. The confidence, especially at the boundaries of the observed secondary structure elements, is found to increase when evaluating 13C chemical shifts, here either by using TALOS or in terms of 13C SCS.

Combining Cell-Free Protein Synthesis and NMR Into a Tool to Study Capsid Assembly Modulation.

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.

100 kHz MAS Proton-Detected NMR Spectroscopy of Hepatitis B Virus Capsids.

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.

Structural Studies of Self-Assembled Subviral Particles: Combining Cell-Free Expression with 110†kHz MAS NMR Spectroscopy.

Viral membrane proteins are prime targets in combatting infection. Still, the determination of their structure remains a challenge, both with respect to sample preparation and the need for structural methods allowing for analysis in a native-like lipid environment. Cell-free protein synthesis and solid-state NMR spectroscopy are promising approaches in this context, the former with respect to its great potential in the native expression of complex proteins, and the latter for the analysis of membrane proteins in lipids. Herein, we show that milligram amounts of the small envelope protein of the duck hepatitis B virus (DHBV) can be produced by cell-free expression, and that the protein self-assembles into subviral particles. Proton-detected 2D NMR spectra recorded at a magic-angle-spinning frequency of 110 kHz on <500 µg protein show a number of isolated peaks with line widths comparable to those of model membrane proteins, paving the way for structural studies of this protein that is homologous to a potential drug target in HBV infection.

Alternative salt bridge formation in Aß-a hallmark of early-onset Alzheimer's disease?

Recently the 3D structure of the Osaka mutant form (E22Δ) of Amyloid-ß1-40 has been determined. We here compare the NMR chemical-shift with the published shifts of a brain-seeded form of wild-type Aß and suggest that the determined mutant fold is accessible to the wild-type protein as well, with small conformational adaptations which accommodate the E22 residue missing in the Osaka mutant. In addition, we illustrate how other mutants could also conform to this model. The stabilization of the N-terminal part of the protein via an intermolecular salt bridge to Lys28 may represent a common structural motif for the mutants which are related to early-onset Alzheimer disease. This feature might connect to the observed increased toxicity of the mutant forms compared to wild-type Aß1-40, where the salt bridge involving Lys28 is intramolecular.