A Biochemical and Biophysical Analysis of the Interaction of nsp9 with nsp12 from SARS-CoV-2-Implications for Future Drug Discovery Efforts.

The ongoing global pandemic of the coronavirus 2019 (COVID-19) disease is caused by the virus SARS-CoV-2, with very few highly effective antiviral treatments currently available. The machinery responsible for the replication and transcription of viral RNA during infection is made up of several important proteins. Two of these are nsp12, the catalytic subunit of the viral polymerase, and nsp9, a cofactor of nsp12 involved in the capping and priming of viral RNA. While several recent studies have determined the structural details of the interaction of nsp9 with nsp12 in the context of RNA capping, very few biochemical or biophysical details are currently available. In this study, we have used a combination of surface plasmon resonance (SPR) experiments, size exclusion chromatography (SEC) experiments, and biochemical assays to identify specific nsp9 residues that are critical for nsp12 binding as well as RNAylation, both of which are essential for the RNA capping process. Our data indicate that nsp9 dimerization is unlikely to play a significant functional role in the virus. We confirm that a set of recently discovered antiviral peptides inhibit nsp9-nsp12 interaction by specifically binding to nsp9; however, we find that these peptides do not impact RNAylation. In summary, our results have important implications for future drug discovery efforts to combat SARS-CoV-2 and any newly emerging coronaviruses.

The oligomeric states of dye-decolorizing peroxidases from Streptomyces lividans and their implications for mechanism of substrate oxidation.

A common evolutionary mechanism in biology to drive function is protein oligomerization. In prokaryotes, the symmetrical assembly of repeating protein units to form homomers is widespread, yet consideration in vitro of whether such assemblies have functional or mechanistic consequences is often overlooked. Dye-decolorizing peroxidases (DyPs) are one such example, where their dimeric α + ß barrel units can form various oligomeric states, but the oligomer influence, if any, on mechanism and function has received little attention. In this work, we have explored the oligomeric state of three DyPs found in Streptomyces lividans, each with very different mechanistic behaviors in their reactions with hydrogen peroxide and organic substrates. Using analytical ultracentrifugation, we reveal that except for one of the A-type DyPs where only a single sedimenting species is detected, oligomer states ranging from homodimers to dodecamers are prevalent in solution. Using cryo-EM on preparations of the B-type DyP, we determined a 3.02 Å resolution structure of a hexamer assembly that corresponds to the dominant oligomeric state in solution as determined by analytical ultracentrifugation. Furthermore, cryo-EM data detected sub-populations of higher-order oligomers, with one of these formed by an arrangement of two B-type DyP hexamers to give a dodecamer assembly. Our solution and structural insights of these oligomer states provide a new framework to consider previous mechanistic studies of these DyP members and are discussed in terms of long-range electron transfer for substrate oxidation and in the "storage" of oxidizable equivalents on the heme until a two-electron donor is available.