Rebecca Jernigan
Arizona State University
ABSTRACT
NendoU (Uridine-specific nidoviral endoribonuclease) is a key drug target from the SARS-CoV-2 virus as NendoU is essential for evasion of the host immune system. While significant advancements have been made in research to fight the COVID-19 pandemic, including vaccine development, therapeutics for treatment are limited. 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) to form the basis for rational drug design. NendoU has been identified to specifically degrade the poly-uridine (poly-U) leader sequences on the negative strand viral RNA. The polyU sequence is recognized by multiple antiviral immune system pathways and their degradation by NendoU results in severely weakened immune response. To fully understand the mechanism for drug discovery and biologic understanding of this unique SARS-CoV-2 protein, 5 and 21 nucleotide substrate RNAs were designed based on sequence analyses of the viral RNA and the catalytic binding pocket. In this poster, we present the first preliminary results of the active NendoU/citrate structure solved to 2.6 Å at room temperature the Linac Coherent Light Source. The structure was refined to an Rwork/Rfree of 0.30/0.36. To advance these studies, mix-and-inject TR-SFX experiments are proposed to capture the catalytic mechanism of NendoU, reveal conformational changes of RNA binding in the NendoU hexamer, and use mutants of NendoU to understand binding and activity differences with new variants of SARS-CoV-2. A mutant of NendoU that tightly bind the substrates but cannot cleave them, H234A, of NendoU will be used to capture the mechanics of RNA docking in early time points with 5nt and 21nt RNA. The active NendoU protein will be used to characterize the intermediate states of the NendoU mechanism and substrate release. Finally, a comparative early time point data set will be collected of a NendoU mutant that recently appeared, migth be hyperactive and is highly prevalent in the California variant of SARS-CoV-2. Understanding the structure and catalytic mechanism of NendoU could lead to optimization of existing drugs and novel drug development for those infected or exposed to the SARS-CoV-2 virus.
Poster Session Link:
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Email: rjjernig@asu.edu