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.

Hepatitis B virus (HBV) replicates its 3 kb DNA genome through capsid-internal reverse transcription, initiated by assembly of 120 core protein (HBc) dimers around a complex of viral pregenomic (pg) RNA and polymerase. Following synthesis of relaxed circular (RC) DNA capsids can be enveloped and secreted as stable virions. Upon infection of a new cell, however, the capsid disintegrates to release the RC-DNA into the nucleus for conversion into covalently closed circular (ccc) DNA. HBc´s interactions with nucleic acids are mediated by an arginine-rich C terminal domain (CTD) with intrinsically strong non-specific RNA binding activity. Adaptation to the changing demands for nucleic acid binding during the viral life cycle is thought to involve dynamic phosphorylation / dephosphorylation events. However, neither the relevant enzymes nor their target sites in HBc are firmly established. Here we developed a bacterial coexpression system enabling access to definably phosphorylated HBc. Combining Phos-tag gel electrophoresis, mass spectrometry and mutagenesis we identified seven of the eight hydroxy amino acids in the CTD as target sites for serine-arginine rich protein kinase 1 (SRPK1); fewer sites were phosphorylated by PKA and PKC. Phosphorylation of all seven sites reduced nonspecific RNA encapsidation as drastically as deletion of the entire CTD and altered CTD surface accessibility, without major structure changes in the capsid shell. The bulk of capsids from human hepatoma cells was similarly highly, yet non-identically, phosphorylated as by SRPK1. While not proving SRPK1 as the infection-relevant HBc kinase the data suggest a mechanism whereby high-level HBc phosphorylation principally suppresses RNA binding whereas one or few strategic dephosphorylation events enable selective packaging of the pgRNA/polymerase complex. The tools developed in this study should greatly facilitate the further deciphering of the role of HBc phosphorylation in HBV infection and its evaluation as a potential new therapeutic target. The liver-pathogenic hepatitis B virus (HBV) is a small enveloped DNA virus that replicates through reverse transcription of a pregenomic (pg)RNA. This requires specific encapsidation of pgRNA and viral polymerase into a shell of 240 core protein (HBc) subunits. Capsid-internal formation of relaxed circular (RC) DNA enables the particle to leave the cell as stable virion; yet, when infecting a new cell it must release the RC-DNA for conversion into another, plasmid-like DNA that templates new viral RNAs. This up and down in nucleic acid interactions is presumably regulated by transient phosphorylation of HBc, mainly in its arginine-rich C terminal domain (CTD) which displays strong non-sequence-specific RNA binding. However, neither the phosphorylation sites nor the relevant enzymes are well defined. We developed a recombinant system to produce kinase-specific phospho-HBc species, and adapted a feasible gel assay for their separation. By mutagenesis and mass spectrometry we identified seven target sites for a major candidate kinase, SRPK1, in the CTD. As full SRPK1 phosphorylation thwarted non-specific RNA binding the comparably high phosphorylation of HBc in human cells suggests how specific pgRNA encapsidation might be achieved. Our new tool set will facilitate disentangling the role of HBc phosphorylation in HBV infection and exploiting it as potential therapeutic target.

Active-site serine D,D-transpeptidases belonging to the penicillin-binding protein family (PBPs) have been considered for a long time as essential for peptidoglycan cross-linking in all bacteria. However, bypass of the PBPs by an L,D-transpeptidase (Ldt_fm) conveys high-level resistance to β-lactams of the penam class in Enterococcus faecium with a minimal inhibitory concentration (MIC) of ampicillin >2,000 µg/ml. Unexpectedly, Ldt_fm does not confer resistance to β-lactams of the carbapenem class (imipenem MIC = 0.5 µg/ml) whereas cephems display residual activity (ceftriaxone MIC = 128 µg/ml). Mass spectrometry, fluorescence kinetics, and NMR chemical shift perturbation experiments were performed to explore the basis for this specificity and identify β-lactam features that are critical for efficient L,D-transpeptidase inactivation. We show that imipenem, ceftriaxone, and ampicillin acylate Ldt_fm by formation of a thioester bond between the active-site cysteine and the β-lactam-ring carbonyl. However, slow acylation and slow acylenzyme hydrolysis resulted in partial Ldt_fm inactivation by ampicillin and ceftriaxone. For ampicillin, Ldt_fm acylation was followed by rupture of the C⁵–C⁶ bond of the β-lactam ring and formation of a secondary acylenzyme prone to hydrolysis. The saturable step of the catalytic cycle was the reversible formation of a tetrahedral intermediate (oxyanion) without significant accumulation of a non-covalent complex. In agreement, a derivative of Ldt_fm blocked in acylation bound ertapenem (a carbapenem), ceftriaxone, and ampicillin with similar low affinities. Thus, oxyanion and acylenzyme stabilization are both critical for rapid L,D-transpeptidase inactivation and antibacterial activity. These results pave the way for optimization of the β-lactam scaffold for L,D-transpeptidase-inactivation.

Significance: Infection with the Rift Valley fever virus (RVFV) has the capacity to cause fatal hemorrhagic fever in humans. A unique characteristic of RVFV infection is the presence of nuclear filaments whose formation is linked to synthesis of the viral NSs protein. We identify a crucial interaction between a ΩX a V motif present in the NSs protein and the p62 subunit of the host TFIIH. This interaction is required for nuclear filament formation, NSs-dependent degradation of p62 and for virulence. This ΩX a V motif is also found in human proteins that bind p62 and our results are an example of how viruses incorporate simple motifs into their protein sequences to mimic human proteins and enhance their functional capabilities in host cells during infections.

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.

P65 is a member of the NF-κB family of transcriptional regulatory proteins that functions as the activating component of the p65–p50 heterodimer. Through its acidic transactivation domain (TAD), p65 has the capacity to form interactions with several different transcriptional regulatory proteins, including TFIIB, TFIIH, CREB-binding protein (CBP)/p300 and TAF_II31. Like other acidic TADs, the p65 TAD contains two subdomains (p65_TA1 and p65_TA2) that interact with different regulatory factors depending on the target gene. Despite its role in controlling numerous NF-κB target genes, there are no high-resolution structures of p65_TA1 bound to a target transcriptional regulatory factor. In this work, we characterize the interaction of p65_TA1 with two factors, the Tfb1/p62 subunit of TFIIH and the KIX domain of CBP. In these complexes, p65_TA1 transitions into a helical conformation that includes its characteristic ΦXXΦΦ motif (Φ = hydrophobic amino acid). Structural and functional studies demonstrate that the two binding interfaces are primarily stabilized by three hydrophobic amino acids within the ΦXXΦΦ motif and these residues are also crucial to its ability to activate transcription. Taken together, the results provide an atomic level description of how p65_TA1 is able to bind different transcriptional regulatory factors needed to activate NF-κB target genes.