ABSTRACT
Prokaryotes have evolved intricate innate immune systems against phage infection1-7. Gabija is a highly widespread prokaryotic defence system that consists of two components, GajA and GajB8. GajA functions as a DNA endonuclease that is inactive in the presence of ATP9. Here, to explore how the Gabija system is activated for anti-phage defence, we report its cryo-electron microscopy structures in five states, including apo GajA, GajA in complex with DNA, GajA bound by ATP, apo GajA-GajB, and GajA-GajB in complex with ATP and Mg2+. GajA is a rhombus-shaped tetramer with its ATPase domain clustered at the centre and the topoisomerase-primase (Toprim) domain located peripherally. ATP binding at the ATPase domain stabilizes the insertion region within the ATPase domain, keeping the Toprim domain in a closed state. Upon ATP depletion by phages, the Toprim domain opens to bind and cleave the DNA substrate. GajB, which docks on GajA, is activated by the cleaved DNA, ultimately leading to prokaryotic cell death. Our study presents a mechanistic landscape of Gabija activation.
Subject(s)
Bacillus cereus , Bacterial Proteins , Bacteriophages , Cryoelectron Microscopy , Immunity, Innate , Adenosine Triphosphatases/metabolism , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/ultrastructure , Adenosine Triphosphate/chemistry , Adenosine Triphosphate/metabolism , Apoproteins/chemistry , Apoproteins/immunology , Apoproteins/metabolism , Apoproteins/ultrastructure , Bacterial Proteins/chemistry , Bacterial Proteins/immunology , Bacterial Proteins/metabolism , Bacterial Proteins/ultrastructure , Bacteriophages/immunology , DNA/metabolism , DNA/chemistry , DNA Cleavage , Magnesium/chemistry , Magnesium/metabolism , Models, Molecular , Protein Binding , Protein Domains , Microbial Viability , Bacillus cereus/chemistry , Bacillus cereus/immunology , Bacillus cereus/metabolism , Bacillus cereus/ultrastructure , Protein Structure, Quaternary , DNA Primase/chemistry , DNA Primase/metabolism , DNA Primase/ultrastructure , DNA Topoisomerases/chemistry , DNA Topoisomerases/metabolism , DNA Topoisomerases/ultrastructureABSTRACT
The noradrenaline transporter has a pivotal role in regulating neurotransmitter balance and is crucial for normal physiology and neurobiology1. Dysfunction of noradrenaline transporter has been implicated in numerous neuropsychiatric diseases, including depression and attention deficit hyperactivity disorder2. Here we report cryo-electron microscopy structures of noradrenaline transporter in apo and substrate-bound forms, and as complexes with six antidepressants. The structures reveal a noradrenaline transporter dimer interface that is mediated predominantly by cholesterol and lipid molecules. The substrate noradrenaline binds deep in the central binding pocket, and its amine group interacts with a conserved aspartate residue. Our structures also provide insight into antidepressant recognition and monoamine transporter selectivity. Together, these findings advance our understanding of noradrenaline transporter regulation and inhibition, and provide templates for designing improved antidepressants to treat neuropsychiatric disorders.