Allosteric regulation and crystallographic fragment screening of SARS-CoV-2 NSP15 endoribonuclease

DOI: 10.1093/nar/gkad314

Authors: Andre Schutzer Godoy (University of Sao Paulo) , Aline Minalli Nakamura (University of Sao Paulo) , Alice Douangamath (Diamond Light Source; Research Complex at Harwell) , Yun Song (Diamond Light Source) , Gabriela Dias Noske (University of Sao Paulo) , Victor Oliveira Gawriljuk (University of Sao Paulo) , Rafaela Sachetto Fernandes (University of Sao Paulo) , Humberto D'Muniz Pereira (University of Sao Paulo) , Ketllyn irene  Zagato Oliveira (University of Sao Paulo) , Daren Fearon (Diamond Light Source; Research Complex at Harwell) , Alexandre Dias (Diamond Light Source; Research Complex at Harwell) , Tobias Krojer (MAX IV Laboratory) , Michael Fairhead (University of Oxford) , Alisa Powell (Diamond Light Source; Research Complex at Harwell) , Louise Dunnett (Diamond Light Source; Research Complex at Harwell) , Jose Brandao-Neto (Diamond Light Source; Research Complex at Harwell) , Rachael Skyner (Diamond Light Source; Research Complex at Harwell) , Rod Chalk (University of Oxford) , Dávid Bajusz (Research Centre for Natural Sciences (Hungary)) , Miklós Bege (University of Debrece) , Anikó Borbás (University of Debrece) , György Miklós Keserű (Research Centre for Natural Sciences (Hungary)) , Frank Von Delft (Diamond Light Source; Research Complex at Harwell; University of Oxford; University of Johannesburg) , Glaucius Oliva (University of Sao Paulo)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Nucleic Acids Research , VOL 579

State: Published (Approved)
Published: April 2023
Diamond Proposal Number(s): 27083 , 27023

Abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). The NSP15 endoribonuclease enzyme, known as NendoU, is highly conserved and plays a critical role in the ability of the virus to evade the immune system. NendoU is a promising target for the development of new antiviral drugs. However, the complexity of the enzyme's structure and kinetics, along with the broad range of recognition sequences and lack of structural complexes, hampers the development of inhibitors. Here, we performed enzymatic characterization of NendoU in its monomeric and hexameric form, showing that hexamers are allosteric enzymes with a positive cooperative index, and with no influence of manganese on enzymatic activity. Through combining cryo-electron microscopy at different pHs, X-ray crystallography and biochemical and structural analysis, we showed that NendoU can shift between open and closed forms, which probably correspond to active and inactive states, respectively. We also explored the possibility of NendoU assembling into larger supramolecular structures and proposed a mechanism for allosteric regulation. In addition, we conducted a large fragment screening campaign against NendoU and identified several new allosteric sites that could be targeted for the development of new inhibitors. Overall, our findings provide insights into the complex structure and function of NendoU and offer new opportunities for the development of inhibitors.

Diamond Keywords: COVID-19; Viruses; Enzymes

Subject Areas: Biology and Bio-materials, Chemistry, Medicine

Diamond Offline Facilities: Electron Bio-Imaging Centre (eBIC), XChem
Instruments: I04-1-Macromolecular Crystallography (fixed wavelength) , Krios II-Titan Krios II at Diamond

Other Facilities: MANACA at LNLS; BioMAX at MAX IV

Added On: 01/05/2023 09:33

Documents:
gkad314.pdf

Discipline Tags:

Pathogens Infectious Diseases Health & Wellbeing Biochemistry Chemistry Structural biology Drug Discovery Life Sciences & Biotech

Technical Tags:

Diffraction Microscopy Fragment Screening Electron Microscopy (EM) Cryo Electron Microscopy (Cryo EM)