TY - JOUR
T1 - Cryo-EM structure of the RADAR supramolecular anti-phage defense complex
AU - Duncan-Lowey, Brianna
AU - Tal, Nitzan
AU - Johnson, Alex G.
AU - Rawson, Shaun
AU - Mayer, Megan L.
AU - Doron, Shany
AU - Millman, Adi
AU - Melamed, Sarah
AU - Fedorenko, Taya
AU - Kacen, Assaf
AU - Brandis, Alexander
AU - Mehlman, Tevie
AU - Amitai, Gil
AU - Sorek, Rotem
AU - Kranzusch, Philip J.
N1 - The authors thank members of the Kranzusch and Sorek laboratories for helpful discussions. We thank W. Shih’s laboratory for training and use of the JEOL-1400 electron microscope, the Harvard Center for Cryo-Electron Microscopy (HC2EM), the HMS Electron Microscopy Facility, M. Eck for sharing computational resources, and the SBGrid consortium for computational support. This study was supported by the Pew Biomedical Scholars Program (P.J.K.), the Burroughs Wellcome Fund PATH award (P.J.K.), the Mathers Foundation (P.J.K.), the Parker Institute for Cancer Immunotherapy (P.J.K.), European Research Council grant ERC-CoG 681203 (R.S.), Israel Science Foundation grant ISF 296/21 (R.S.), the Ernest and Bonnie Beutler Research Program of Excellence in Genomic Medicine (R.S.), the Minerva Foundation and Federal German Ministry for Education and Research (R.S.), the Knell Family Center for Microbiology (R.S.), the Yotam project and the Weizmann Institute Sustainability and Energy Research Initiative (R.S.), and the Dr. Barry Sherman Institute for Medicinal Chemistry (R.S.). B.D.-L. was supported by a Herchel Smith Graduate Research Fellowship, A.G.J. by a Life Science Research Foundation postdoctoral fellowship of the Open Philanthropy Project, and A.M. by a fellowship from the Ariane de Rothschild Women Doctoral Program and, in part, by the Israeli Council for Higher Education via the Weizmann Data Science Research Center. Author contributions - Experiments were designed by B.D.-L., N.T., R.S., and P.J.K. Protein purification, biochemical experiments, and molecular modeling were performed by B.D.-L., with assistance from P.J.K. Phage challenge assays were performed by N.T., T.F., and S.M. Cryo-EM data were collected by A.G.J. and M.L.M., with assistance from S.R. Cryo-EM data processing was performed by S.R. Cell lysate extraction and analysis of MS data was performed by N.T. Genome analyses were performed by S.D., A.K., and G.A. RNA-seq was performed by N.T., and RNA-seq data analysis was performed by A.M. and N.T. Quantitative mass spectrometry was performed by A.B. and T.M. The manuscript was written by B.D.-L., N.T., R.S., and P.J.K., and all authors contributed to editing the manuscript and support the conclusions.
PY - 2023/3/2
Y1 - 2023/3/2
N2 - RADAR is a two-protein bacterial defense system that was reported to defend against phage by “editing” messenger RNA. Here, we determine cryo-EM structures of the RADAR defense complex, revealing RdrA as a heptameric, two-layered AAA+ ATPase and RdrB as a dodecameric, hollow complex with twelve surface-exposed deaminase active sites. RdrA and RdrB join to form a giant assembly up to 10 MDa, with RdrA docked as a funnel over the RdrB active site. Surprisingly, our structures reveal an RdrB active site that targets mononucleotides. We show that RdrB catalyzes ATP-to-ITP conversion in vitro and induces the massive accumulation of inosine mononucleotides during phage infection in vivo, limiting phage replication. Our results define ATP mononucleotide deamination as a determinant of RADAR immunity and reveal supramolecular assembly of a nucleotide-modifying machine as a mechanism of anti-phage defense.
AB - RADAR is a two-protein bacterial defense system that was reported to defend against phage by “editing” messenger RNA. Here, we determine cryo-EM structures of the RADAR defense complex, revealing RdrA as a heptameric, two-layered AAA+ ATPase and RdrB as a dodecameric, hollow complex with twelve surface-exposed deaminase active sites. RdrA and RdrB join to form a giant assembly up to 10 MDa, with RdrA docked as a funnel over the RdrB active site. Surprisingly, our structures reveal an RdrB active site that targets mononucleotides. We show that RdrB catalyzes ATP-to-ITP conversion in vitro and induces the massive accumulation of inosine mononucleotides during phage infection in vivo, limiting phage replication. Our results define ATP mononucleotide deamination as a determinant of RADAR immunity and reveal supramolecular assembly of a nucleotide-modifying machine as a mechanism of anti-phage defense.
UR - http://www.scopus.com/inward/record.url?scp=85149068771&partnerID=8YFLogxK
U2 - https://doi.org/10.1016/j.cell.2023.01.012
DO - https://doi.org/10.1016/j.cell.2023.01.012
M3 - مقالة
C2 - 36764290
SN - 0092-8674
VL - 186
SP - 987
EP - 998
JO - Cell
JF - Cell
IS - 5
ER -