John Kirkpatrick

Knowledge of site-specific dynamics of biomolecules is necessary to understand their specific functions. Solid-state NMR is a uniquely powerful technique that can provide both structural and motional information about complex biomolecules. In recent years, nucleic acids have become increasingly studied by ssNMR, but as yet few RNA or DNA structures are available, with ssNMR-derived dynamics data on RNA rarer still. Here, we report the first systematic ssNMR study of ¹⁵ N T ₁ relaxation in RNA using straightforward nucleotide-type-specific and uniform labeling schemes. We observe clear correlation of the measured ¹⁵ N T ₁ relaxation time-constants with different structural elements in the RNA molecule, reflecting the distinct characteristics of their underlying motions. We anticipate that this novel approach can be further developed to provide a detailed and comprehensive picture of RNA dynamics in large biomolecular machines.

Non-ribosomal peptide synthetases (NRPSs) are macromolecular enzymatic complexes responsible for the biosynthesis of an array of microbial natural products, many of which have important applications for human health. The nature of the NRPS machinery, which has been likened to an assembly line, should be amenable to bio-engineering efforts directed towards efficient synthesis of novel and tailored molecules. However, the success of such endeavours depends on a detailed understanding of the mechanistic principles governing the various steps in the peptide assembly-line. Here, we report the near-complete assignment of the Ile, Met, Leu and Val methyl-groups of the 59-kDa adaptor-condensation di-domain (BN-BC) from the Tomaymycin NRPS. These assignments will provide the foundation for future NMR studies of the complex dynamic behaviour of the condensation domain both in isolation and in the context of the enzymatic cycle, which will themselves form the basis for developing a complete mechanistic description of the central condensation reaction in this prototypical NRPS.

In the quest for new bioactive substances, nonribosomal peptide synthetases (NRPS) provide biodiversity by synthesizing nonproteinaceous peptides with high cellular activity. NRPS machinery consists of multiple modules, each catalyzing a unique series of chemical reactions. Incomplete understanding of the biophysical principles orchestrating these reaction arrays limits the exploitation of NRPSs in synthetic biology. Here, we use nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry to solve the conundrum of how intermodular recognition is coupled with loaded carrier protein specificity in the tomaymycin NRPS. We discover an adaptor domain that directly recruits the loaded carrier protein from the initiation module to the elongation module and reveal its mechanism of action. The adaptor domain of the type found here has specificity rules that could potentially be exploited in the design of engineered NRPS machinery.

We have determined the heterogeneous structural ensemble of the tandem SH2 domains of the protein tyrosine phosphatase SHP2 in agreement with experimental data from small-angle X-ray scattering and NMR residual dipolar couplings in solution.

Knowledge of RNA structure, either in isolation or in complex, is fundamental to understand the mechanism of cellular processes. Solid‐state NMR (ssNMR) is applicable to high molecular‐weight complexes and does not require crystallization; thus, it is well‐suited to study RNA as part of large multicomponent assemblies. Recently, we solved the first structures of both RNA and an RNA‐protein complex by ssNMR using conventional ¹³C‐ and ¹⁵N‐detection. This approach is limited by the severe overlap of the RNA peaks together with the low sensitivity of multidimensional experiments. Here, we overcome the limitations in sensitivity and resolution by using ¹H‐detection at fast MAS rates. We develop experiments that allow the identification of complete nucleobase spin‐systems together with their site‐specific base pair pattern using sub‐milligram quantities of one uniformly labelled RNA sample. These experiments provide rapid access to RNA secondary structure by ssNMR in protein‐RNA complexes of any size.

Die Aufklärung von RNA Strukturen, entweder in Isolation oder im Komplex, ist essentiell, um die Mechanismen zellulärer Prozesse zu verstehen. Festkörper‐NMR ist auf Komplexe mit hohem Molekulargewicht anwendbar und benötigt keine Kristallisation; Die Methode ist daher gut geeignet um RNA als Teil von großen mehrkomponentigen Komplexen zu untersuchen. Kürzlich konnten wir die ersten Strukturen von RNA und einem RNA‐Protein Komplex mittels Festkörper‐NMR durch die Anwendung von konventioneller ¹³C‐ und ¹⁵N‐Detektion lösen. Dieser Ansatz ist durch diverse RNA Peak‐Überlappungen und die niedrige Intensität von multidimensionalen Experimenten limitiert. In dieser Arbeit überwinden wir die Limitationen in Sensitivität und Auflösung durch die Anwendung von ¹H‐Detektion bei schnellen MAS Geschwindigkeiten. Wir entwickeln Experimente, die die Identifizierung von vollständigen Spinsystemen der Nukleobasen zusammen mit derer Nukleotid‐spezifischer Basenpaarung ermöglichen. Diese Experimente benötigen weniger als ein Milligramm einer uniform Isotopen‐markierten RNA Probe und erlauben die schnelle Bestimmung der RNA Sekundärstruktur durch Festkörper‐NMR in Protein‐RNA Komplexen jedweder Größe.

The current COVID-19 pandemic caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has become a worldwide health crisis, necessitating coordinated scientific research and urgent identification of new drug targets for treatment of COVID-19 lung disease. The covid19-nmr consortium seeks to support drug development by providing publicly accessible NMR data on the viral RNA elements and proteins. The SARS-CoV-2 genome comprises a single RNA of about 30 kb in length, in which 14 open reading frames (ORFs) have been annotated, and encodes approximately 30 proteins. The first two-thirds of the SARS-CoV-2 genome is made up of two large overlapping open-reading-frames (ORF1a and ORF1b) encoding a replicase polyprotein, which is subsequently cleaved to yield 16 so-called non-structural proteins. The non-structural protein 1 (Nsp1), which is considered to be a major virulence factor, suppresses host immune functions by associating with host ribosomal complexes at the very end of its C-terminus. Furthermore, Nsp1 facilitates initiation of viral RNA translation via an interaction of its N-terminal domain with the 5′ untranslated region (UTR) of the viral RNA. Here, we report the near-complete backbone chemical-shift assignments of full-length SARS-CoV-2 Nsp1 (19.8 kDa), which reveal the domain organization, secondary structure and backbone dynamics of Nsp1, and which will be of value to further NMR-based investigations of both the biochemical and physiological functions of Nsp1.

Histone deacetylases (HDACs) are key enzymes in epigenetics and important drug targets in cancer biology. Whilst it has been established that HDACs regulate many cellular processes, far less is known about the regulation of these enzymes themselves. Here, we show that HDAC8 is allosterically regulated by shifts in populations between exchanging states. An inactive state is identified, which is stabilised by a range of mutations and resembles a sparsely-populated state in equilibrium with active HDAC8. Computational models show that the inactive and active states differ by small changes in a regulatory region that extends up to 28 Å from the active site. The regulatory allosteric region identified here in HDAC8 corresponds to regions in other class I HDACs known to bind regulators, thus suggesting a general mechanism. The presented results pave the way for the development of allosteric HDAC inhibitors and regulators to improve the therapy for several disease states.