Molecular basis of Gabija anti-phage supramolecular assemblies

As one of the most prevalent anti-phage defense systems in prokaryotes, Gabija consists of a Gabija protein A (GajA) and a Gabija protein B (GajB). The assembly and function of the Gabija system remain unclear. Here we present cryo-EM structures of GajA and the GajAB complex, revealing tetrameric and octameric assemblies, respectively. In the center of the complex, GajA assembles into a symmetric tetramer, which recruits two sets of GajB dimer at opposite sides of the complex, resulting in a 4:4 GajAB supramolecular complex for anti-phage defense. Further biochemical analysis showed that GajA alone is sufficient to cut double-stranded DNA and plasmid DNA, which can be inhibited by ATP. Unexpectedly, the GajAB displays enhanced activity for plasmid DNA, suggesting a role of substrate selection by GajB. Together, our study defines a framework for understanding anti-phage immune defense by the GajAB complex.

[1]  Feng-Ting Huang,et al.  Prokaryotic Gabija complex senses and executes nucleotide depletion and DNA cleavage for antiviral defense , 2023, bioRxiv.

[2]  Yina Gao,et al.  Molecular basis of RADAR anti-phage supramolecular assemblies , 2023, Cell.

[3]  Gil Amitai,et al.  Cryo-EM structure of the RADAR supramolecular anti-phage defense complex , 2022, Cell.

[4]  E. Koonin,et al.  Prokaryotic innate immunity through pattern recognition of conserved viral proteins , 2022, Science.

[5]  Gil Amitai,et al.  An expanding arsenal of immune systems that protect bacteria from phages , 2022, bioRxiv.

[6]  R. Sorek,et al.  SnapShot: Bacterial immunity , 2022, Cell.

[7]  P. J. Kranzusch,et al.  CBASS phage defense and evolution of antiviral nucleotide signaling. , 2022, Current opinion in immunology.

[8]  M. Touchon,et al.  Systematic and quantitative view of the antiviral arsenal of prokaryotes , 2021, Nature Communications.

[9]  Oriol Vinyals,et al.  Highly accurate protein structure prediction with AlphaFold , 2021, Nature.

[10]  Feng-Ting Huang,et al.  A nucleotide-sensing endonuclease from the Gabija bacterial defense system , 2020, bioRxiv.

[11]  Conrad C. Huang,et al.  UCSF ChimeraX: Structure visualization for researchers, educators, and developers , 2020, Protein science : a publication of the Protein Society.

[12]  Jonathan L. Schmid-Burgk,et al.  Diverse enzymatic activities mediate antiviral immunity in prokaryotes , 2020, Science.

[13]  J. Chappie,et al.  The full-length structure of Thermus scotoductus OLD defines the ATP hydrolysis properties and catalytic mechanism of Class 1 OLD family nucleases , 2020, Nucleic acids research.

[14]  J. Chappie,et al.  Structural characterization of Class 2 OLD family nucleases supports a two-metal catalysis mechanism for cleavage , 2019, Nucleic acids research.

[15]  Rotem Sorek,et al.  Systematic discovery of antiphage defense systems in the microbial pangenome , 2018, Science.

[16]  Eugene V Koonin,et al.  Diversity, classification and evolution of CRISPR-Cas systems. , 2017, Current opinion in microbiology.

[17]  David J. Fleet,et al.  cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination , 2017, Nature Methods.

[18]  O. Song,et al.  ATP‐dependent DNA binding, unwinding, and resection by the Mre11/Rad50 complex , 2016, The EMBO journal.

[19]  Wei Yang,et al.  UvrD Helicase Unwinds DNA One Base Pair at a Time by a Two-Part Power Stroke , 2006, Cell.

[20]  Conrad C. Huang,et al.  UCSF Chimera—A visualization system for exploratory research and analysis , 2004, J. Comput. Chem..

[21]  R. Henderson,et al.  Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. , 2003, Journal of molecular biology.

[22]  John A. Tainer,et al.  Structural Biology of Rad50 ATPase ATP-Driven Conformational Control in DNA Double-Strand Break Repair and the ABC-ATPase Superfamily , 2000, Cell.

[23]  S. Velankar,et al.  Crystal Structures of Complexes of PcrA DNA Helicase with a DNA Substrate Indicate an Inchworm Mechanism , 1999, Cell.

[24]  Detlef D. Leipe,et al.  Toprim--a conserved catalytic domain in type IA and II topoisomerases, DnaG-type primases, OLD family nucleases and RecR proteins. , 1998, Nucleic acids research.

[25]  Gabriel Waksman,et al.  Major Domain Swiveling Revealed by the Crystal Structures of Complexes of E. coli Rep Helicase Bound to Single-Stranded DNA and ADP , 1997, Cell.

[26]  Christopher J. Williams,et al.  MolProbity: More and better reference data for improved all‐atom structure validation , 2018, Protein science : a publication of the Protein Society.

[27]  E. Lingohr,et al.  Enumeration of bacteriophages using the small drop plaque assay system. , 2009, Methods in molecular biology.

[28]  E. Lingohr,et al.  Enumeration of bacteriophages by double agar overlay plaque assay. , 2009, Methods in molecular biology.

[29]  W. Delano.  The PyMOL Molecular Graphics System , 2002 .