The assembly platform FimD is required to obtain the most stable quaternary structure of type 1 pili.

Type 1 pili are important virulence factors of uropathogenic Escherichia coli that mediate bacterial attachment to epithelial cells in the urinary tract. The pilus rod is comprised of thousands of copies of the main structural subunit FimA and is assembled in vivo by the assembly platform FimD. Although type 1 pilus rods can self-assemble from FimA in vitro, this reaction is slower and produces structures with lower kinetic stability against denaturants compared to in vivo-assembled rods. Our study reveals that FimD-catalysed in vitro-assembled type 1 pilus rods attain a similar stability as pilus rods assembled in vivo. Employing structural, biophysical and biochemical analyses, we show that in vitro assembly reactions lacking FimD produce pilus rods with structural defects, reducing their stability against dissociation. Overall, our results indicate that FimD is not only required for the catalysis of pilus assembly, but also to control the assembly of the most stable quaternary structure.

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.

High and fast: NMR protein-proton side-chain assignments at 160 kHz and 1.2 GHz.

The NMR spectra of side-chain protons in proteins provide important information, not only about their structure and dynamics, but also about the mechanisms that regulate interactions between macromolecules. However, in the solid-state, these resonances are particularly difficult to resolve, even in relatively small proteins. We show that magic-angle-spinning (MAS) frequencies of 160 kHz, combined with a high magnetic field of 1200 MHz proton Larmor frequency, significantly improve their spectral resolution. We investigate in detail the gain for MAS frequencies between 110 and 160 kHz MAS for a model sample as well as for the hepatitis B viral capsid assembled from 120 core-protein (Cp) dimers. For both systems, we found a significantly improved spectral resolution of the side-chain region in the 1H-13C 2D spectra. The combination of 160 kHz MAS frequency with a magnetic field of 1200 MHz, allowed us to assign 61% of the aliphatic protons of Cp. The side-chain proton assignment opens up new possibilities for structural studies and further characterization of protein-protein or protein-nucleic acid interactions.

Structural conservation of HBV-like capsid proteins over hundreds of millions of years despite the shift from non-enveloped to enveloped life-style.

The discovery of nackednaviruses provided new insight into the evolutionary history of the hepatitis B virus (HBV): The common ancestor of HBV and nackednaviruses was non-enveloped and while HBV acquired an envelope during evolution, nackednaviruses remained non-enveloped. We report the capsid structure of the African cichlid nackednavirus (ACNDV), determined by cryo-EM at 3.7 Å resolution. This enables direct comparison with the known capsid structures of HBV and duck HBV, prototypic representatives of the mammalian and avian lineages of the enveloped Hepadnaviridae, respectively. The sequence identity with HBV is 24% and both the ACNDV capsid protein fold and the capsid architecture are very similar to those of the Hepadnaviridae and HBV in particular. Acquisition of the hepadnaviral envelope was thus not accompanied by a major change in capsid structure. Dynamic residues at the spike tip are tentatively assigned by solid-state NMR, while the C-terminal domain is invisible due to dynamics. Solid-state NMR characterization of the capsid structure reveals few conformational differences between the quasi-equivalent subunits of the ACNDV capsid and an overall higher capsid structural disorder compared to HBV. Despite these differences, the capsids of ACNDV and HBV are structurally highly similar despite the 400 million years since their separation.

Molecular elucidation of drug-induced abnormal assemblies of the hepatitis B virus capsid protein by solid-state NMR.

Hepatitis B virus (HBV) capsid assembly modulators (CAMs) represent a recent class of anti-HBV antivirals. CAMs disturb proper nucleocapsid assembly, by inducing formation of either aberrant assemblies (CAM-A) or of apparently normal but genome-less empty capsids (CAM-E). Classical structural approaches have revealed the CAM binding sites on the capsid protein (Cp), but conformational information on the CAM-induced off-path aberrant assemblies is lacking. Here we show that solid-state NMR can provide such information, including for wild-type full-length Cp183, and we find that in these assemblies, the asymmetric unit comprises a single Cp molecule rather than the four quasi-equivalent conformers typical for the icosahedral T = 4 symmetry of the normal HBV capsids. Furthermore, while in contrast to truncated Cp149, full-length Cp183 assemblies appear, on the mesoscopic level, unaffected by CAM-A, NMR reveals that on the molecular level, Cp183 assemblies are equally aberrant. Finally, we use a eukaryotic cell-free system to reveal how CAMs modulate capsid-RNA interactions and capsid phosphorylation. Our results establish a structural view on assembly modulation of the HBV capsid, and they provide a rationale for recently observed differences between in-cell versus in vitro capsid assembly modulation.

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 Structure of Amyloid-ß Fibrils.

Amyloid fibrils are involved in a number of diseases and notably play a role in neurodegeneration, where they are present in plaques in the brain. Their structure determination might help in finding ways to interfere with their formation, and ultimately prevent disease, by revealing the structure-function relationship and helping to design molecules targeting initial assembly steps and further propagation. Here, we describe the different steps in NMR protocols which allowed the 3D structure determination of amyloid-ß fibrils.

α-Synuclein Fibril, Ribbon and Fibril-91 Amyloid Polymorphs Generation for Structural Studies.

The human α-synuclein protein, identified as one of the main markers of Parkinson's disease, is a 140-amino acid thermostable protein that can easily be overexpressed in E. coli. The purification protocol determines the ability of the protein to assemble into amyloid fibrils of well-defined structures. Here, we describe the purification and assembly protocols to obtain three well-characterized amyloid forms (ribbon, fibrils, and fibril-91) used to assess their activity in biochemical and cellular assays or to investigate their atomic structure by cryo-electron microscopy and solid-state NMR.

Pharmacomodulation of a ligand targeting the HBV capsid hydrophobic pocket.

Hepatitis B virus (HBV) is a small enveloped retrotranscribing DNA virus and an important human pathogen. Its capsid-forming core protein (Cp) features a hydrophobic pocket proposed to be central notably in capsid envelopment. Indeed, mutations in and around this pocket can profoundly modulate, and even abolish, secretion of enveloped virions. We have recently shown that Triton X-100, a detergent used during Cp purification, binds to the hydrophobic pocket with micromolar affinity. We here performed pharmacomodulation of pocket binders through systematic modifications of the three distinct chemical moieties composing the Triton X-100 molecule. Using NMR and ITC, we found that the flat aromatic moiety is essential for binding, while the number of atoms of the aliphatic chain modulates binding affinity. The hydrophilic tail, in contrast, is highly tolerant to changes in both length and type. Our data provide essential information for designing a new class of HBV antivirals targeting capsid-envelope interactions.

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.

Correction of field instabilities in biomolecular solid-state NMR by simultaneous acquisition of a frequency reference.

With the advent of faster magic-angle spinning (MAS) and higher magnetic fields, the resolution of biomolecular solid-state nuclear magnetic resonance (NMR) spectra has been continuously increasing. As a direct consequence, the always narrower spectral lines, especially in proton-detected spectroscopy, are also becoming more sensitive to temporal instabilities of the magnetic field in the sample volume. Field drifts in the order of tenths of parts per million occur after probe insertion or temperature change, during cryogen refill, or are intrinsic to the superconducting high-field magnets, particularly in the months after charging. As an alternative to a field-frequency lock based on deuterium solvent resonance rarely available for solid-state NMR, we present a strategy to compensate non-linear field drifts using simultaneous acquisition of a frequency reference (SAFR). It is based on the acquisition of an auxiliary 1D spectrum in each scan of the experiment. Typically, a small-flip-angle pulse is added at the beginning of the pulse sequence. Based on the frequency of the maximum of the solvent signal, the field evolution in time is reconstructed and used to correct the raw data after acquisition, thereby acting in its principle as a digital lock system. The general applicability of our approach is demonstrated on 2D and 3D protein spectra during various situations with a non-linear field drift. SAFR with small-flip-angle pulses causes no significant loss in sensitivity or increase in experimental time in protein spectroscopy. The correction leads to the possibility of recording high-quality spectra in a typical biomolecular experiment even during non-linear field changes in the order of 0.1 ppm h-1 without the need for hardware solutions, such as stabilizing the temperature of the magnet bore. The improvement of linewidths and peak shapes turns out to be especially important for 1H-detected spectra under fast MAS, but the method is suitable for the detection of carbon or other nuclei as well.

Spectroscopic glimpses of the transition state of ATP hydrolysis trapped in a bacterial DnaB helicase.

The ATP hydrolysis transition state of motor proteins is a weakly populated protein state that can be stabilized and investigated by replacing ATP with chemical mimics. We present atomic-level structural and dynamic insights on a state created by ADP aluminum fluoride binding to the bacterial DnaB helicase from Helicobacter pylori. We determined the positioning of the metal ion cofactor within the active site using electron paramagnetic resonance, and identified the protein protons coordinating to the phosphate groups of ADP and DNA using proton-detected 31P,1H solid-state nuclear magnetic resonance spectroscopy at fast magic-angle spinning > 100 kHz, as well as temperature-dependent proton chemical-shift values to prove their engagements in hydrogen bonds. 19F and 27Al MAS NMR spectra reveal a highly mobile, fast-rotating aluminum fluoride unit pointing to the capture of a late ATP hydrolysis transition state in which the phosphoryl unit is already detached from the arginine and lysine fingers.

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.

Temperature-Dependent Solid-State NMR Proton Chemical-Shift Values and Hydrogen Bonding.

Temperature-dependent NMR experiments are often complicated by rather long magnetic-field equilibration times, for example, occurring upon a change of sample temperature. We demonstrate that the fast temporal stabilization of a magnetic field can be achieved by actively stabilizing the temperature of the magnet bore, which allows quantification of the weak temperature dependence of a proton chemical shift, which can be diagnostic for the presence of hydrogen bonds. Hydrogen bonding plays a central role in molecular recognition events from both fields, chemistry and biology. Their direct detection by standard structure-determination techniques, such as X-ray crystallography or cryo-electron microscopy, remains challenging due to the difficulties of approaching the required resolution, on the order of 1 Å. We, herein, explore a spectroscopic approach using solid-state NMR to identify protons engaged in hydrogen bonds and explore the measurement of proton chemical-shift temperature coefficients. Using the examples of a phosphorylated amino acid and the protein ubiquitin, we show that fast magic-angle spinning (MAS) experiments at 100 kHz yield sufficient resolution in proton-detected spectra to quantify the rather small chemical-shift changes upon temperature variations.

A rationally designed oral vaccine induces immunoglobulin A in the murine gut that directs the evolution of attenuated Salmonella variants.

The ability of gut bacterial pathogens to escape immunity by antigenic variation-particularly via changes to surface-exposed antigens-is a major barrier to immune clearance1. However, not all variants are equally fit in all environments2,3. It should therefore be possible to exploit such immune escape mechanisms to direct an evolutionary trade-off. Here, we demonstrate this phenomenon using Salmonella enterica subspecies enterica serovar Typhimurium (S.Tm). A dominant surface antigen of S.Tm is its O-antigen: a long, repetitive glycan that can be rapidly varied by mutations in biosynthetic pathways or by phase variation4,5. We quantified the selective advantage of O-antigen variants in the presence and absence of O-antigen-specific immunoglobulin A and identified a set of evolutionary trajectories allowing immune escape without an associated fitness cost in naive mice. Through the use of rationally designed oral vaccines, we induced immunoglobulin A responses blocking all of these trajectories. This selected for Salmonella mutants carrying deletions of the O-antigen polymerase gene wzyB. Due to their short O-antigen, these evolved mutants were more susceptible to environmental stressors (detergents or complement) and predation (bacteriophages) and were impaired in gut colonization and virulence in mice. Therefore, a rationally induced cocktail of intestinal antibodies can direct an evolutionary trade-off in S.Tm. This lays the foundations for the exploration of mucosal vaccines capable of setting evolutionary traps as a prophylactic strategy.

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.

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.

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.