Room-temperature structural studies of SARS-CoV-2 protein NendoU with an X-ray free-electron laser

Rebecca J Jernigan; Dhenugen Logeswaran; Diandra Doppler; Nirupa Nagaratnam; Mukul Sonker; Jay-How Yang; Gihan Ketawala; Jose M Martin-Garcia; Megan L Shelby; Thomas D Grant; Valerio Mariani; Alexandra Tolstikova; Michelle Z Sheikh; Mimi Cho Yung; Matthew A Coleman; Sahba Zaare; Emily K Kaschner; Mohammad Towshif Rabbani; Reza Nazari; Michele A Zacks; Brandon Hayes; Raymond G Sierra; Mark S Hunter; Stella Lisova; Alexander Batyuk; Christopher Kupitz; Sebastien Boutet; Debra T Hansen; Richard A Kirian; Marius Schmidt; Raimund Fromme; Matthias Frank; Alexandra Ros; Julian J-L Chen; Sabine Botha; Petra Fromme

doi:10.1016/j.str.2022.12.009

Room-temperature structural studies of SARS-CoV-2 protein NendoU with an X-ray free-electron laser

Structure. 2023 Feb 2;31(2):138-151.e5. doi: 10.1016/j.str.2022.12.009. Epub 2023 Jan 10.

Authors

Rebecca J Jernigan¹, Dhenugen Logeswaran², Diandra Doppler¹, Nirupa Nagaratnam³, Mukul Sonker¹, Jay-How Yang³, Gihan Ketawala¹, Jose M Martin-Garcia³, Megan L Shelby⁴, Thomas D Grant⁵, Valerio Mariani⁶, Alexandra Tolstikova⁷, Michelle Z Sheikh³, Mimi Cho Yung⁴, Matthew A Coleman⁴, Sahba Zaare⁸, Emily K Kaschner¹, Mohammad Towshif Rabbani³, Reza Nazari³, Michele A Zacks³, Brandon Hayes⁶, Raymond G Sierra⁶, Mark S Hunter⁶, Stella Lisova⁶, Alexander Batyuk⁶, Christopher Kupitz⁶, Sebastien Boutet⁶, Debra T Hansen³, Richard A Kirian⁹, Marius Schmidt¹⁰, Raimund Fromme¹, Matthias Frank⁴, Alexandra Ros¹, Julian J-L Chen², Sabine Botha¹¹, Petra Fromme¹²

Affiliations

¹ Biodesign Center for Applied Structural Discovery, Arizona State University, Tempe, AZ 85287-5001, USA; School of Molecular Sciences, Arizona State University, Tempe, AZ 85287-1604, USA.
² School of Molecular Sciences, Arizona State University, Tempe, AZ 85287-1604, USA.
³ Biodesign Center for Applied Structural Discovery, Arizona State University, Tempe, AZ 85287-5001, USA.
⁴ Lawrence Livermore National Laboratory, 7000 East Avenue, Livermore, CA 94550, USA.
⁵ Department of Structural Biology, Jacobs School of Medicine and Biomedical Sciences, SUNY University at Buffalo, 955 Main Street, Buffalo, NY 14203, USA.
⁶ Linac Coherent Light Source, SLAC National Accelerator Laboratory, Menlo Park, CA 94025, USA.
⁷ Deutsches Elektronen-Synchrotron, Notkestrasse 85, 22607 Hamburg, Germany.
⁸ Biodesign Center for Applied Structural Discovery, Arizona State University, Tempe, AZ 85287-5001, USA; Fulton School of Electrical, Computer, and Energy Engineering, Arizona State University, Tempe, AZ 85287, USA; Department of Physics, Arizona State University, Tempe, AZ 85287-1504, USA.
⁹ Biodesign Center for Applied Structural Discovery, Arizona State University, Tempe, AZ 85287-5001, USA; Department of Physics, Arizona State University, Tempe, AZ 85287-1504, USA.
¹⁰ Department of Physics, University of Wisconsin-Milwaukee, 3135 N. Maryland Avenue, Milwaukee, WI 53211, USA.
¹¹ Biodesign Center for Applied Structural Discovery, Arizona State University, Tempe, AZ 85287-5001, USA; Department of Physics, Arizona State University, Tempe, AZ 85287-1504, USA. Electronic address: sbotha@asu.edu.
¹² Biodesign Center for Applied Structural Discovery, Arizona State University, Tempe, AZ 85287-5001, USA; School of Molecular Sciences, Arizona State University, Tempe, AZ 85287-1604, USA. Electronic address: pfromme@asu.edu.

Abstract

NendoU from SARS-CoV-2 is responsible for the virus's ability to evade the innate immune system by cleaving the polyuridine leader sequence of antisense viral RNA. Here we report the room-temperature structure of NendoU, solved by serial femtosecond crystallography at an X-ray free-electron laser to 2.6 Å resolution. The room-temperature structure provides insight into the flexibility, dynamics, and other intrinsic properties of NendoU, with indications that the enzyme functions as an allosteric switch. Functional studies examining cleavage specificity in solution and in crystals support the uridine-purine cleavage preference, and we demonstrate that enzyme activity is fully maintained in crystal form. Optimizing the purification of NendoU and identifying suitable crystallization conditions set the benchmark for future time-resolved serial femtosecond crystallography studies. This could advance the design of antivirals with higher efficacy in treating coronaviral infections, since drugs that block allosteric conformational changes are less prone to drug resistance.

Keywords: COVID-19; NSP15; NendoU; SARS-CoV-2; X-ray free-electron laser; serial femtosecond crystallography.

Publication types

Research Support, Non-U.S. Gov't
Research Support, N.I.H., Extramural
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

COVID-19*
Crystallography, X-Ray
Electrons
Humans
Lasers
SARS-CoV-2*
Temperature

Substances

nidoviral uridylate-specific endoribonuclease

Abstract

Publication types

MeSH terms

Substances

Grants and funding