Rebecca Jernigan

Structural and functional studies of prophylactic drug target, NendoU, from SARS-CoV-2 for time-resolved serial femtosecond crystallography and rational drug design

Rebecca Jernigan, Arizona State University, Graduate Student

Nonstructural protein 15 (Nsp15) is a Uridine specific nidoviral endoribonuclease (often called NendoU) from the SARS-CoV-2 virus, the causative agent of the COVID-19 pandemic. NendoU aides in the evasion of the host immune system by degrading the poly-uridine (Poly-U) leader sequence on the anti-sense viral RNA. Loss of activity in NendoU would leave the Poly-U leaders intact, which has been shown to have an earlier and more robust immune response, making NendoU an attractive prophylactic drug target against COVID-19.The goal of the project is to determine the first snapshots of NendoU's catalytic mechanism by time-resolved serial femtosecond (fs) crystallography (TR-SFX). In this poster, we present the first results of the active NendoU/citrate structure solved to 2.6 degrees at room temperature at the Linac Coherent Light Source (LCLS) X-ray Free Electron Laser (XFEL) at the Stanford Linear Accelerator Center (SLAC). The H234A inactive mutant for RNA docking was solved to 4.7, also under SFX conditions at LCLS. These experiments demonstrate successful diffraction of NendoU microcrystals at LCLS and structural analysis reveals that the active site is well positioned in the solvent channel to allow for diffusion of the enzyme. Additionally, RNA substrate (5 and 21 nucleotides) was designed using analysis of the NendoU active site and sequence analysis of the viral RNA substrate. NendoU microcrystals were shown to remain active and cleave the RNA substrate, required for proposed TR-SFX experiments. In addition to the new structural and functional information provided in this studies, the exact conditions for performing TR-SFX were established. Using microfluidic mix-and-inject devices, NendoU microcrystals will be mixed with RNA substrate at millisecond time points. The ultra fast time points can only be solved using the brilliance and timescales achieved by an XFEL, making the experimentation process extremely competitive. By solving the structure at different time points, we hope to assemble a 'molecular movie' that directly observes substrate binding, cleavage, and release. This information would be a major advance in understanding the biology of NendoU as well as identifying important conformational changes and sites that could be used to inhibit the structural mechanism, and therefore its function of evading the coronavirus infection from the host immune system. Optimization of existing drugs and novel drug development would benefit those infected or exposed to the SARS-CoV-2 virus and would provide an additional direction for COVID-19 treatment, not previously used. Because NendoU is highly conserved across the Coronaviridae family, it also is promising for new variants and future coronaviruses, as it is not very prone to large or rapid changes.

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