Presentation Information
[P03-317]Structural Basis for DinG Helicases as a Central Immune Scaffold in Prokaryotic Argonautes Mediated Host Defense
○Tong Wu1 (1. Shanghai Jiao Tong University (China))
Keywords:
DinG helicases,pAgo nucleases,prokaryotic host defense,recruitment module,immune scaffold
[Purpose] Prokaryotes utilize sophisticated defense systems to neutralize invasive mobile genetic elements (MGEs). Recent studies have highlighted the significant potential of prokaryotic Argonautes (pAgos) in prokaryotic host defense. We observed that a specific class of pAgo is located within the same gene neighborhood as the helicase DinG, suggesting that DinG may play a crucial role in supporting pAgo-mediated elimination of foreign mobile genetic elements. DinG, a member of the helicase superfamily 2 (SF2), has been traditionally implicated in maintaining genomic stability through the resolution of R-loop and D-loop structures. Through structural determination, we aim to uncover how DinG coordinates with its cognate pAgo effector, a synergy that is essential for constructing an indispensable immune barrier against exogenous genetic elements.
[Method] Size-exclusion chromatography (SEC); cryo-electron microscopy (cryo-EM); AlphaFold 3 (AF3) computational modeling.
[Results] We determined the high-resolution structure of DinG using cryo-EM. The structure confirms that DinG belongs to the helicase superfamily 2 and possesses characteristic domains required for nucleic acid binding and ATP hydrolysis. While DinG retains its ancestral biochemical capacity to resolve R-loop and D-loop structures, our structural analysis reveals an evolutionarily specialized structural module within its functional domains dedicated to protein-protein interactions. To understand the coordination between DinG and its immune effectors, we utilized AF3 to simulate the assembly of DinG with its syntenic pAgo partner. The AF3 modeling revealed a high-confidence and evolutionarily conserved docking interface between the specialized module in the DinG structural domains and the pAgo nuclease. This structural arrangement indicates that the interface is essential for the recruitment of pAgo, and suggests that DinG may regulate pAgo’s catalytic activity. Beyond its role as a scaffold, we identified that DinG possesses intrinsic nuclease activity, allowing it to degrade DNA in a non-specific manner. Our experimental evidence demonstrates that the formation of the DinG-pAgo complex significantly accelerates the degradation rate of foreign nucleic acids compared to the individual proteins. In the presence of both guide DNA (gDNA) and target DNA (tDNA), DinG and pAgo function in concert to facilitate rapid cleavage. Conversely, when the interaction is disrupted, both proteins exhibit markedly diminished degradation capacity. These findings underscore the functional necessity of the DinG-pAgo association for the efficient neutralization of MGEs and a rapid host defense response.
[Consideration] These findings mark a paradigm shift in our understanding of SF2 helicases. The determined structure of DinG provides the molecular basis for its transition from a repair factor to an immune scaffold, illustrating how conserved helicases are functionally repurposed within prokaryotic immunity. The integration of a helicase motor with nuclease effectors likely facilitates the efficient degradation of invasive foreign genetic elements.
[Conclusion] This work establishes DinG as a core immune scaffold rather than a generic DNA maintenance enzyme. By defining the high-resolution structure of DinG and the interaction landscape of its recruitment module, this study offers new insights into the evolutionary trajectory of helicases toward specialized defense systems.
[Method] Size-exclusion chromatography (SEC); cryo-electron microscopy (cryo-EM); AlphaFold 3 (AF3) computational modeling.
[Results] We determined the high-resolution structure of DinG using cryo-EM. The structure confirms that DinG belongs to the helicase superfamily 2 and possesses characteristic domains required for nucleic acid binding and ATP hydrolysis. While DinG retains its ancestral biochemical capacity to resolve R-loop and D-loop structures, our structural analysis reveals an evolutionarily specialized structural module within its functional domains dedicated to protein-protein interactions. To understand the coordination between DinG and its immune effectors, we utilized AF3 to simulate the assembly of DinG with its syntenic pAgo partner. The AF3 modeling revealed a high-confidence and evolutionarily conserved docking interface between the specialized module in the DinG structural domains and the pAgo nuclease. This structural arrangement indicates that the interface is essential for the recruitment of pAgo, and suggests that DinG may regulate pAgo’s catalytic activity. Beyond its role as a scaffold, we identified that DinG possesses intrinsic nuclease activity, allowing it to degrade DNA in a non-specific manner. Our experimental evidence demonstrates that the formation of the DinG-pAgo complex significantly accelerates the degradation rate of foreign nucleic acids compared to the individual proteins. In the presence of both guide DNA (gDNA) and target DNA (tDNA), DinG and pAgo function in concert to facilitate rapid cleavage. Conversely, when the interaction is disrupted, both proteins exhibit markedly diminished degradation capacity. These findings underscore the functional necessity of the DinG-pAgo association for the efficient neutralization of MGEs and a rapid host defense response.
[Consideration] These findings mark a paradigm shift in our understanding of SF2 helicases. The determined structure of DinG provides the molecular basis for its transition from a repair factor to an immune scaffold, illustrating how conserved helicases are functionally repurposed within prokaryotic immunity. The integration of a helicase motor with nuclease effectors likely facilitates the efficient degradation of invasive foreign genetic elements.
[Conclusion] This work establishes DinG as a core immune scaffold rather than a generic DNA maintenance enzyme. By defining the high-resolution structure of DinG and the interaction landscape of its recruitment module, this study offers new insights into the evolutionary trajectory of helicases toward specialized defense systems.
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