@article{d764de696b064add98557a3c5364022a,
title = "Room-temperature structural studies of SARS-CoV-2 protein NendoU with an X-ray free-electron laser",
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 {\AA} 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",
author = "Jernigan, {Rebecca J.} and Dhenugen Logeswaran and Diandra Doppler and Nirupa Nagaratnam and Mukul Sonker and Yang, {Jay How} and Gihan Ketawala and Martin-Garcia, {Jose M.} and Shelby, {Megan L.} and Grant, {Thomas D.} and Valerio Mariani and Alexandra Tolstikova and Sheikh, {Michelle Z.} and Yung, {Mimi Cho} and Coleman, {Matthew A.} and Sahba Zaare and Kaschner, {Emily K.} and Rabbani, {Mohammad Towshif} and Reza Nazari and Zacks, {Michele A.} and Brandon Hayes and Sierra, {Raymond G.} and Hunter, {Mark S.} and Stella Lisova and Alexander Batyuk and Christopher Kupitz and Sebastien Boutet and Hansen, {Debra T.} and Kirian, {Richard A.} and Marius Schmidt and Raimund Fromme and Matthias Frank and Alexandra Ros and Chen, {Julian J.L.} and Sabine Botha and Petra Fromme",
note = "Funding Information: We are very grateful for all the people who supported the experiment at LCLS by being present in person and participating remotely during the onset of the COVID-19 pandemic in August 2020. We acknowledge LCLS at SLAC for the provision of RAPID ACCESS X-ray free-electron laser beamtime at the MFX scientific instrument and would like to thank all staff members of LCLS for their strong support. We would further like to thank Robin Stanley and Meredith Frazier for providing access to their cryo-EM maps. We also thank Oleksandr Yefanov for providing detector geometry files. This work was supported by two RAPID grant awards from the National Science Foundation, “IIBR: Instrumentation: Time-resolved studies of the SARS-CoV-2 endonuclease NP15” (award 2031343) and ”Structure, function and dynamics of SARS Coronavirus-2 main protease 3CLpro determined with mix-and-inject serial XFEL crystallography” (award 2030466). The work was also supported by the NSF Science and Technology Center award “Biology with X-ray lasers (BioXFEL)” (award 1231306). D.L. was supported by NIH grant R01GM094450. J.M.M-G. was funded by the Community of Madrid through an “Atracci{\'o}n y retenci{\'o}n de talento” grant (2019-T1/BMD-15552). The work was also supported by funds from National Institutes of Health grants R01GM095583 and 5R01GM117342, by the Biodesign Center for Applied Structural Discovery at ASU, and the US Department of Energy through Lawrence Livermore National Laboratory under contract DE-AC52-07NA27344. Use of the LCLS, SLAC National Accelerator Laboratory, is supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under contract DE-AC02-76SF00515. Experimental design, P.F. S. Botha, A.R. and J.J-L.C.; in vitro expression and purification, M.L.S. M.A.C. and M.F.; in vivo expression, R.J.J. M.Z.S. E.K.K. N.N. and M.C.Y.; plasmid design, D.T.H. and M.C.Y.; in vitro purification, R.J.J. J-H.Y. N.N. and M.Z.S.; crystallization, J.M.M-G. R.J.J. P.F. N.N. and D.L.; analysis of the RNA sequences and design of the RNA substrates, J.J-L.C.; RNA assays, D.L. J.J-L.C. N.N. and R.J.J.; XFEL data collection at LCLS, R.J.J. P.F. D.D. M. Sonker, B.H. R.G.S. M.S.H. S.L. A.B. C.K. S. Boutet, S. Botha, G.K. T.D.G. V.M. A.T. R.F. M. Schmidt, M.F. and M.A.Z.; microfluidics/injection, D.D. M. Sonker, M.T.R. S.L. R.N. R.A.K. and R.G.S.; beamline operation, B.H. R.G.S. M.S.H. S.L. A.B. C.K. and S. Boutet; XFEL data analysis, S. Botha, G.K. T.D.G. V.M. A.T. and M. Schmidt; writing and editing of the manuscript, all authors. The authors declare no competing interests. We support inclusive, diverse, and equitable conduct of research. Funding Information: This work was supported by two RAPID grant awards from the National Science Foundation , “IIBR: Instrumentation: Time-resolved studies of the SARS-CoV-2 endonuclease NP15” (award 2031343) and ”Structure, function and dynamics of SARS Coronavirus-2 main protease 3CLpro determined with mix-and-inject serial XFEL crystallography” (award 2030466). The work was also supported by the NSF Science and Technology Center award “Biology with X-ray lasers (BioXFEL)” (award 1231306). D.L. was supported by NIH grant R01GM094450 . J.M.M-G. was funded by the Community of Madrid through an “ Atracci{\'o}n y retenci{\'o}n de talento ” grant ( 2019-T1/BMD-15552 ). The work was also supported by funds from National Institutes of Health grants R01GM095583 and 5R01GM117342 , by the Biodesign Center for Applied Structural Discovery at ASU , and the US Department of Energy through Lawrence Livermore National Laboratory under contract DE-AC52-07NA27344 . Use of the LCLS, SLAC National Accelerator Laboratory, is supported by the US Department of Energy , Office of Science , Office of Basic Energy Sciences under contract DE-AC02-76SF00515 . Publisher Copyright: {\textcopyright} 2022",
year = "2023",
month = feb,
day = "2",
doi = "10.1016/j.str.2022.12.009",
language = "English (US)",
volume = "31",
pages = "138--151.e5",
journal = "Structure with Folding & design",
issn = "0969-2126",
publisher = "Cell Press",
number = "2",
}