PA4608 is a single PilZ domain protein from Pseudomonas aeruginosa that binds to cyclic dimeric guanosine monophosphate (c-di-GMP). Although the monomeric structure of unbound PA4608 has been studied in detail, the molecular details of c-di-GMP binding to this protein are still uncharacterized. Hence, we determined the solution structure of c-di-GMP bound PA4608. We found that PA4608 undergoes conformational changes to expose the c-di-GMP binding site by ejection of the C-terminal 3 10 helix. A dislocation of the C-terminal tail in the presence of c-di-GMP implies that this region acts as a lid that alternately covers and exposes the hydrophobic surface of the binding site. In addition, mutagenesis and NOE data for PA4608 revealed that conserved residues are in contact with the c-di-GMP molecule. The unique structural characteristics of PA4608, including its monomeric state and its ligand binding characteristics, yield insight into its function as a c-di-GMP receptor.
Cyclic GMP-AMP synthase (cGAS) is a DNA-sensing enzyme in the innate immune system. Recent studies using core-cGAS lacking the N terminus investigated the mechanism for binding of double-stranded (ds) DNA and synthesis of 2',3'-cyclic GMP-AMP (cGAMP), a secondary messenger that ultimately induces type I interferons. However, the function of the N terminus of cGAS remains largely unknown. Here, we found that the N terminus enhanced the activity of core-cGAS in vivo. Importantly, the catalytic activity of core-cGAS decreased as the length of double-stranded DNA (dsDNA) increased, but the diminished activity was restored by addition of the N terminus. Furthermore, the N terminus de-oligomerized the 2 : 2 complex of core-cGAS and dsDNA into a 1 : 1 complex, suggesting that the N terminus enhanced the activity of core-cGAS by facilitating formation of a monomeric complex of cGAS and DNA.
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