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Structure-guided reprogramming of human cGAS dinucleotide linkage specificity.

Philip J Kranzusch1, Amy S Y Lee2, Stephen C Wilson3

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This study reveals how bacterial enzymes and human cGAS synthesize cyclic dinucleotides (CDNs) with specific linkages. Understanding this mechanism clarifies bacterial pathogenesis and innate immunity pathways.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Immunology

Background:

  • Cyclic dinucleotides (CDNs) are crucial signaling molecules in bacterial pathogenesis and mammalian innate immunity.
  • The enzyme cGAS produces 2'-5' cGAMP, essential for immune stimulation, but its linkage specificity is not understood.
  • Prokaryotic enzymes with similar functions to cGAS are largely uncharacterized.

Purpose of the Study:

  • To elucidate the molecular mechanism underlying the 2'-5' phosphodiester linkage specificity in cGAS-mediated cyclic dinucleotide synthesis.
  • To investigate the structure and function of the bacterial enzyme DncV as a prokaryotic homolog of cGAS.
  • To establish mechanistic homology between bacterial signaling pathways and mammalian innate immunity.

Main Methods:

  • High-resolution crystal structures of Vibrio cholerae DncV and human cGAS.
  • Biochemical assays to analyze CDN synthesis and linkage specificity.
  • Site-directed mutagenesis to reprogram the human cGAS active site and cellular assays to test STING activation.

Main Results:

  • DncV, a prokaryotic enzyme, shares mechanistic similarities with human cGAS in cyclic dinucleotide synthesis.
  • Crystal structures reveal that DncV and cGAS synthesize CDNs via sequential reactions proceeding in opposite directions.
  • Reprogramming the human cGAS active site enabled the production of 3'-5' cGAMP, selectively activating alternative STING adaptor alleles.

Conclusions:

  • The active site configuration of cGAS and DncV dictates the specific phosphodiester linkage chemistry (2'-5' or 3'-5') of cyclic dinucleotides.
  • This linkage specificity controls downstream signaling pathways in both bacterial and mammalian systems.
  • Mechanistic homology between bacterial pathogenesis factors and innate immune enzymes provides insights into conserved signaling mechanisms.