RESUMEN
Coronaviruses modify their single-stranded RNA genome with a methylated cap during replication to mimic the eukaryotic mRNAs. The capping process is initiated by several nonstructural proteins (nsp) encoded in the viral genome. The methylation is performed by two methyltransferases, nsp14 and nsp16, while nsp10 acts as a co-factor to both. Additionally, nsp14 carries an exonuclease domain which operates in the proofreading system during RNA replication of the viral genome. Both nsp14 and nsp16 were reported to independently bind nsp10, but the available structural information suggests that the concomitant interaction between these three proteins would be impossible due to steric clashes. Here, we show that nsp14, nsp10, and nsp16 can form a heterotrimer complex upon significant allosteric change. This interaction is expected to encourage the formation of mature capped viral mRNA, modulating nsp14's exonuclease activity, and protecting the viral RNA. Our findings show that nsp14 is amenable to allosteric regulation and may serve as a novel target for therapeutic approaches.
Asunto(s)
Metiltransferasas , ARN Viral , SARS-CoV-2 , Proteínas no Estructurales Virales , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Proteínas no Estructurales Virales/metabolismo , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/química , Metiltransferasas/metabolismo , Metiltransferasas/genética , Metiltransferasas/química , Metilación , ARN Viral/metabolismo , ARN Viral/química , ARN Viral/genética , Exorribonucleasas/metabolismo , Exorribonucleasas/genética , Humanos , Unión Proteica , Caperuzas de ARN/metabolismo , Caperuzas de ARN/genética , Regulación Alostérica , COVID-19/virología , COVID-19/genética , Multimerización de Proteína , Replicación Viral/genética , ARN Mensajero/metabolismo , ARN Mensajero/genética , ARN Mensajero/química , Proteínas Reguladoras y Accesorias ViralesRESUMEN
X-ray free-electron laser (XFEL) light sources have enabled the rapid growth of time-resolved structural experiments, which provide crucial information on the function of macromolecules and their mechanisms. Here, the aim was to commission the SwissMX fixed-target sample-delivery system at the SwissFEL Cristallina experimental station using the PSI-developed micro-structured polymer (MISP) chip for pump-probe time-resolved experiments. To characterize the system, crystals of the light-sensitive protein light-oxygen-voltage domain 1 (LOV1) from Chlamydomonas reinhardtii were used. Using different experimental settings, the accidental illumination, referred to as light contamination, of crystals mounted in wells adjacent to those illuminated by the pump laser was examined. It was crucial to control the light scattering from and through the solid supports otherwise significant contamination occurred. However, the results here show that the opaque MISP chips are suitable for defined pump-probe studies of a light-sensitive protein. The experiment also probed the sub-millisecond structural dynamics of LOV1 and indicated that at Δt = 10â µs a covalent thioether bond is established between reactive Cys57 and its flavin mononucleotide cofactor. This experiment validates the crystals to be suitable for in-depth follow-up studies of this still poorly understood signal-transduction mechanism. Importantly, the fixed-target delivery system also permitted a tenfold reduction in protein sample consumption compared with the more common high-viscosity extrusion-based delivery system. This development creates the prospect of an increase in XFEL project throughput for the field.