A Small Helical Bundle Prepares Primer Synthesis by Binding Two Nucleotides that Enhance Sequence-Specific Recognition of the DNA Template.

Primases have a fundamental role in DNA replication. They synthesize a primer that is then extended by DNA polymerases. Archaeoeukaryotic primases require for synthesis a catalytic and an accessory domain, the exact contribution of the latter being unresolved. For the pRN1 archaeal primase, this domain is a 115-amino acid helix bundle domain (HBD). Our structural investigations of this small HBD by liquid- and solid-state nuclear magnetic resonance (NMR) revealed that only the HBD binds the DNA template. DNA binding becomes sequence-specific after a major allosteric change in the HBD, triggered by the binding of two nucleotide triphosphates. The spatial proximity of the two nucleotides and the DNA template in the quaternary structure of the HBD strongly suggests that this small domain brings together the substrates to prepare the first catalytic step of primer synthesis. This efficient mechanism is likely general for all archaeoeukaryotic primases.

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.

Tony W. Keller (1937-2023).

Tony Keller, a pioneer in the field of Nuclear Magnetic Resonance (NMR) spectroscopy, passed away on October 27, 2023, at the age of 86 in Spiez, Switzerland. His work and vision were essential to the development and commercialization of NMR spectrometers for many areas of scientific research.

Probing a Hydrogen-π Interaction Involving a Trapped Water Molecule in the Solid State.

The detection and characterization of trapped water molecules in chemical entities and biomacromolecules remains a challenging task for solid materials. We herein present proton-detected solid-state Nuclear Magnetic Resonance (NMR) experiments at 100 kHz magic-angle spinning and at high static magnetic-field strengths (28.2 T) enabling the detection of a single water molecule fixed in the calix[4]arene cavity of a lanthanide complex by a combination of three types of non-covalent interactions. The water proton resonances are detected at a chemical-shift value close to zero ppm, which we further confirm by quantum-chemical calculations. Density Functional Theory calculations pinpoint to the sensitivity of the proton chemical-shift value for hydrogen-π interactions. Our study highlights how proton-detected solid-state NMR is turning into the method-of-choice in probing weak non-covalent interactions driving a whole branch of molecular-recognition events in chemistry and biology.

Solid-State NMR Reveals Asymmetric ATP Hydrolysis in the Multidrug ABC Transporter BmrA.

The detailed mechanism of ATP hydrolysis in ATP-binding cassette (ABC) transporters is still not fully understood. Here, we employed 31P solid-state NMR to probe the conformational changes and dynamics during the catalytic cycle by locking the multidrug ABC transporter BmrA in prehydrolytic, transition, and posthydrolytic states, using a combination of mutants and ATP analogues. The 31P spectra reveal that ATP binds strongly in the prehydrolytic state to both ATP-binding sites as inferred from the analysis of the nonhydrolytic E504A mutant. In the transition state of wild-type BmrA, the symmetry of the dimer is broken and only a single site is tightly bound to ADP:Mg2+:vanadate, while the second site is more 'open' allowing exchange with the nucleotides in the solvent. In the posthydrolytic state, weak binding, as characterized by chemical exchange with free ADP and by asymmetric 31P-31P two-dimensional (2D) correlation spectra, is observed for both sites. Revisiting the 13C spectra in light of these findings confirms the conformational nonequivalence of the two nucleotide-binding sites in the transition state. Our results show that following ATP binding, the symmetry of the ATP-binding sites of BmrA is lost in the ATP-hydrolysis step, but is then recovered in the posthydrolytic ADP-bound state.

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.

Emerging Structural Understanding of Amyloid Fibrils by Solid-State NMR.

Amyloid structures at atomic resolution have remained elusive mainly because of their extensive polymorphism and because their polymeric properties have hampered structural studies by classical approaches. Progress in sample preparation, as well as solid-state NMR methods, recently enabled the determination of high-resolution 3D structures of fibrils such as the amyloid-ß fibril, which is involved in Alzheimer's disease. Notably, the simultaneous but independent structure determination of Aß1-42, a peptide that forms fibrillar deposits in the brain of Alzheimer patients, by two independent laboratories, which yielded virtually identical results, has highlighted how structures can be obtained that allow further functional investigation.

How wide is the window opened by high-resolution relaxometry on the internal dynamics of proteins in solution?

The dynamics of molecules in solution is usually quantified by the determination of timescale-specific amplitudes of motions. High-resolution nuclear magnetic resonance (NMR) relaxometry experiments-where the sample is transferred to low fields for longitudinal (T1) relaxation, and back to high field for detection with residue-specific resolution-seeks to increase the ability to distinguish the contributions from motion on timescales slower than a few nanoseconds. However, tumbling of a molecule in solution masks some of these motions. Therefore, we investigate to what extent relaxometry improves timescale resolution, using the "detector" analysis of dynamics. Here, we demonstrate improvements in the characterization of internal dynamics of methyl-bearing side chains by carbon-13 relaxometry in the small protein ubiquitin. We show that relaxometry data leads to better information about nanosecond motions as compared to high-field relaxation data only. Our calculations show that gains from relaxometry are greater with increasing correlation time of rotational diffusion.

Biomolecular solid-state NMR spectroscopy at 1200 MHz: the gain in resolution.

Progress in NMR in general and in biomolecular applications in particular is driven by increasing magnetic-field strengths leading to improved resolution and sensitivity of the NMR spectra. Recently, persistent superconducting magnets at a magnetic field strength (magnetic induction) of 28.2 T corresponding to 1200 MHz proton resonance frequency became commercially available. We present here a collection of high-field NMR spectra of a variety of proteins, including molecular machines, membrane proteins, viral capsids, fibrils and large molecular assemblies. We show this large panel in order to provide an overview over a range of representative systems under study, rather than a single best performing model system. We discuss both carbon-13 and proton-detected experiments, and show that in 13C spectra substantially higher numbers of peaks can be resolved compared to 850 MHz while for 1H spectra the most impressive increase in resolution is observed for aliphatic side-chain resonances.

Paramagnetic Solid-State NMR to Localize the Metal-Ion Cofactor in an Oligomeric DnaB Helicase.

Paramagnetic metal ions can be inserted into ATP-fueled motor proteins by exchanging the diamagnetic Mg2+ cofactor with Mn2+ or Co2+ . Then, paramagnetic relaxation enhancement (PRE) or pseudo-contact shifts (PCSs) can be measured to report on the localization of the metal ion within the protein. We determine the metal position in the oligomeric bacterial DnaB helicase from Helicobacter pylori complexed with the transition-state ATP-analogue ADP:AlF4 - and single-stranded DNA using solid-state NMR and a structure-calculation protocol employing CYANA. We discuss and compare the use of Mn2+ and Co2+ in localizing the ATP cofactor in large oligomeric protein assemblies. 31 P PCSs induced in the Co2+ -containing sample are then used to localize the DNA phosphate groups on the Co2+ PCS tensor surface enabling structural insights into DNA binding to the DnaB helicase.

Insight into small molecule binding to the neonatal Fc receptor by X-ray crystallography and 100 kHz magic-angle-spinning NMR.

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 ß2-microglobulin (ß2m) (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 ß2m, 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.

Dimer Organization of Membrane-Associated NS5A of Hepatitis†C Virus as Determined by Highly Sensitive 1 H-Detected Solid-State NMR.

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.

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.

Proton-Detected Solid-State NMR of the Cell-Free Synthesized α-Helical Transmembrane Protein NS4B from Hepatitis C Virus.

Proton-detected 100 kHz magic-angle-spinning (MAS) solid-state NMR is an emerging analysis method for proteins with only hundreds of microgram quantities, and thus allows structural investigation of eukaryotic membrane proteins. This is the case for the cell-free synthesized hepatitis C virus (HCV) nonstructural membrane protein 4B (NS4B). We demonstrate NS4B sample optimization using fast reconstitution schemes that enable lipid-environment screening directly by NMR. 2D spectra and relaxation properties guide the choice of the best sample preparation to record 2D 1 H-detected 1 H,15 N and 3D 1 H,13 C,15 N correlation experiments with linewidths and sensitivity suitable to initiate sequential assignments. Amino-acid-selectively labeled NS4B can be readily obtained using cell-free synthesis, opening the door to combinatorial labeling approaches which should enable structural studies.

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.

ATP Analogues for Structural Investigations: Case Studies of a DnaB Helicase and an ABC Transporter.

Nucleoside triphosphates (NTPs) are used as chemical energy source in a variety of cell systems. Structural snapshots along the NTP hydrolysis reaction coordinate are typically obtained by adding stable, nonhydrolyzable adenosine triphosphate (ATP) -analogues to the proteins, with the goal to arrest a state that mimics as closely as possible a physiologically relevant state, e.g., the pre-hydrolytic, transition and post-hydrolytic states. We here present the lessons learned on two distinct ATPases on the best use and unexpected pitfalls observed for different analogues. The proteins investigated are the bacterial DnaB helicase from Helicobacter pylori and the multidrug ATP binding cassette (ABC) transporter BmrA from Bacillus subtilis, both belonging to the same division of P-loop fold NTPases. We review the magnetic-resonance strategies which can be of use to probe the binding of the ATP-mimics, and present carbon-13, phosphorus-31, and vanadium-51 solid-state nuclear magnetic resonance (NMR) spectra of the proteins or the bound molecules to unravel conformational and dynamic changes upon binding of the ATP-mimics. Electron paramagnetic resonance (EPR), and in particular W-band electron-electron double resonance (ELDOR)-detected NMR, is of complementary use to assess binding of vanadate. We discuss which analogues best mimic the different hydrolysis states for the DnaB helicase and the ABC transporter BmrA. These might be relevant also to structural and functional studies of other NTPases.

Spinning faster: protein NMR at MAS frequencies up to 126 kHz.

We report linewidth and proton T1, T1ρ and T2' relaxation data of the model protein ubiquitin acquired at MAS frequencies up to 126 kHz. We find a predominantly linear improvement in linewidths and coherence decay times of protons with increasing spinning frequency in the range from 93 to 126 kHz. We further attempt to gain insight into the different contributions to the linewidth at fast MAS using site-specific analysis of proton relaxation parameters and present bulk relaxation times as a function of the MAS frequency. For microcrystalline fully-protonated ubiquitin, inhomogeneous contributions are only a minor part of the proton linewidth, and at 126 kHz MAS coherent effects are still dominating. We furthermore present site-specific proton relaxation rate constants during a spinlock at 126 kHz MAS, as well as MAS-dependent bulk T1ρ (1HN).

A Substantial Structural Conversion of the Native Monomer Leads to in-Register Parallel Amyloid Fibril Formation in Light-Chain Amyloidosis.

Amyloid light-chain (AL) amyloidosis is a rare disease in which plasma-cell-produced monoclonal immunoglobulin light chains misfold and become deposited as fibrils in the extracellular matrix. λ6 subgroup light chains are particularly fibrillogenic, and around 25 % of amyloid-associated λ6 light chains exist as the allotypic G24R variant that renders the protein less stable. The molecular details of this process, as well as the structures of the fibrils, are unknown. We have used solid-state NMR to investigate different fibril polymorphs. The secondary structures derived from NMR predominantly show ß-strands, including in former turn or helical regions, and provide a molecular basis for previously identified fibrillogenic hotspots. We have determined, by using differentially 15 N:13 C-labeled samples, that the ß-strands are stacked in-register parallel in the fibrils. This supramolecular arrangement shows that the native globular folds rearrange substantially upon fibrillization, and rules out the previously hypothesized fibril formation from native monomers.

Characterization of H2 -Splitting Products of Frustrated Lewis Pairs: Benefit of Fast Magic-Angle Spinning.

Proton spectroscopy in solid-state NMR on catalytic materials offers new opportunities in structural characterization, in particular of reaction products of catalytic reactions such as hydrogenation reactions. Unfortunately, the 1 H NMR line widths in magic-angle spinning solid-state spectra are often broadened by an incomplete averaging of 1 H-1 H dipolar couplings. We herein discuss two model compounds, namely the H2 -splitting products of two phosphane-borane Frustrated Lewis Pairs (FLPs), to study potentials and limitations of proton solid-state NMR experiments employing magic-angle spinning frequencies larger than 100 kHz at a static magnetic field strength of 20.0 T. The 1 H lines are homogeneously broadened as illustrated by spin-echo decay experiments. We study two structurally similar materials which however show significant differences in 1 H line widths which we explain by differences in their 1 H-1 H dipolar networks. We discuss the benefit of fast MAS experiments up to 110 kHz to detect the resonances of the H+ /H- pair in the hydrogenation products of FLPs.

Quantifying proton NMR coherent linewidth in proteins under fast MAS conditions: a second moment approach.

Proton detected solid-state NMR under fast magic-angle-spinning (MAS) conditions is currently redefining the applications of solid-state NMR, in particular in structural biology. Understanding the contributions to the spectral linewidth is thereby of paramount importance. When disregarding the sample-dependent inhomogeneous contributions, the NMR proton linewidth is defined by homogeneous broadening, which has incoherent and coherent contributions. Understanding and disentangling these different contributions in multi-spin systems like proteins is still an open issue. The coherent contribution is mainly caused by the dipolar interaction under MAS and is determined by the molecular structure and the proton chemical shifts. Numerical simulation approaches based on numerically exact direct integration of the Liouville-von Neumann equation can give valuable information about the lineshape, but are limited to small spin systems (<12 spins). We present an alternative simulation method for the coherent contributions based on the rapid and partially analytic calculation of the second moments of large spin systems. We first validate the method on a simple system by predicting the 19F linewidth in CaF2 under MAS. We compare simulation results to experimental data for microcrystalline ubiquitin (deuterated 100% back-exchanged at 110 kHz and fully-protonated at 125 kHz). Our results quantitatively explain the observed linewidth per-residue basis for the vast majority of residues.