Fisher, Z. | Search bioRxiv

bioRxiv2023

Oligomeric state of β-coronavirus non-structural protein 10 stimulators studied by OmniSEC and Small Angle X-ray Scattering

Members of the {beta}-coronavirus family such as SARS-CoV-2, SARS, and MERS have caused pandemics over the last 20 years. Future pandemics are likely and studying the coronavirus family members is necessary for their understanding and treatment. Coronaviruses possess 16 non-structural proteins, many of which are involved in viral replication and other vital functions. Non-structural protein 10 (nsp10) is an essential stimulator of nsp14 and nsp16, modulating RNA proofreading and viral RNA cap formation. Studying nsp10 of pathogenic coronaviruses is central to understanding its multifunctional role. We report the biochemical and biophysical characterisation of full-length nsp10 from MERS, SARS and SARS-CoV-2. Proteins were subjected to a combination of OmniSEC and SEC-MALS to characterise their oligomeric state. Full-length nsp10s were predominantly monomeric in solution, while truncated versions of nsp10 have a higher tendency to oligomerise. Small angle X-ray scattering (SAXS) experiments reveal a globular shape of nsp10 which is conserved in all three coronaviruses, including MERS nsp10, which diverges most from SARS and SARS-CoV-2 nsp10s. In conclusion, unbound nsp10 proteins from SARS, MERS, and SARS-CoV-2 are globular and predominantly monomeric in solution. Additionally, we describe for the first time a functional role of the C-terminus of nsp10 for tight binding to nsp14.

biochemistry↗

bioRxiv2020

Visualizing drug inhibitor binding interactions using microcrystal electron diffraction

Visualizing drug inhibitor binding interactions at the atomic level is important for both structure-based drug design and fragment-based screening methods. Rapid and uniform soaking with potentially less lattice defects make small macromolecular crystals attractive targets for studying ligand biding using 3D microcrystal electron diffraction (MicroED). However, so far no drug inhibitor binding interactions could unambiguously be resolved by electron diffraction. Here, we use MicroED to study the binding of a sulfonamide inhibitor to human carbonic anhydrase isoform II (HCA II). We show that MicroED data can efficiently be collected in-house on a conventional TEM from thin hydrated microcrystals after a brief soaking with the clinical drug inhibitor acetazolamide (AZM). The data are of high enough quality to unequivocally fit and resolve the inhibitor bound to the active site of the protein. We anticipate MicroED can play an important role in future drug discovery experiments, complementing existing methods in structural biology such as x-ray and neutron diffraction.

molecular biology↗