| The host uses complex and diverse defense systems to defend itself against outside pathogens.Recently,it has been found a new defense system—cyclic oligonucleotide-based phage signaling system(CBASS),which can prevent phage proliferation by initiating individual cell suicide,thus protecting the entire bacterial population.cGAS/DncV-like nucleotidyltransferase(CD-NTase)is an important component of the CBASS system and belongs to a class of immune-sensing enzymes that synthesize nucleotide second messengers.With the further research,CD-NTases are found in bacteria and mammalian cells.Bacterial CDNTases can activate cascade system and regulate downstream responses to initiate initial antiviral responses.There are more than 5,600 CD-NTases found in bacteria,and they use four nucleotides to synthesize multiple cyclic nucleotides,predicting as many as 180 signal molecules could be synthesized.In animal cells,cGAS is activated by DNA and synthesize 2’3’-cGAMP,which subsequently activates STING and ultimately triggers the release of typeⅠinterferon.The function and catalytic mechanism of cGAS have been elucidated.However,the regulatory mechanism of CD-NTases in bacteria is still unclear and needs to be further elucidated.In this thesis,we selected AtCdnG,which belongs to G cyclase of CD-NTases family as the research object.The AtCdnG enzyme contains two domains,namely the N-terminal domain and the C-terminal domain.The N-terminal domain is the classical nucleotidyltransferase(NTase)domain,which is the catalytic core of the enzyme.It can bind to substrates and catalyze the synthesis of cyclic nucleotides.The C-terminal domain are Adenylyl/Guanylyl and SMODS C-terminal sensor domain(AGS-C),which is predicted to be involved in nucleotide recognition,but its specific function remains unknown.The full-length and truncated proteins of AtCdnG were obtained for crystal search through a series of experiments of expression and purification.Finally,the high-resolution structures of AtCdnG N-terminal domain(1-349,AtCdnGΔC)and the AtCdnGΔC-ATP complex were obtained,with resolutions of 2.23 (?) and 2.4 (?),respectively..Structural analysis showed that the structure of AtCdnΔC adopts al extended bilobed architecture characterized by a nucleotidyltransferase fold.The front-end α/β core and the rearend helical bundle are connected by a long α-helix(spine).The α/β core contains six helices located on one side and a twisted seven-stranded β sheet.The C-terminal helix bundle consists of three helices and one pair of anti-parallel β sheets.The results of DALI search showed that the proteins with similar structure to AtCdnGΔC belong to CD-NTase family,and the most similar protein was B.diazoefficiens CdnG(BpCdnG)(PDB number 7LJN,Z score 27.5),with the classic V.cholerae DncV(PDB number 4U03,Z score 24.0)is also similar.The position of α7(residues:137-150),α9(residues:172-182)and α17(residues:249-261)in the AtCdnGΔC structure were found to be significantly different from those of BdCdnG and DncV,this may be related to the inactive state of AtCdnGΔC and its different catalytic properties with other CD-NTase enzymes.The enzyme activity of AtCdnG was characterized by biochemical experiments.The results showed that AtCdnG could only catalyze ATP reaction in vitro,which indicated that AtCdnG had high substrate preference for ATP.In addition,AtCdnG catalyzes a variety of ATP products in vitro,including cyclic adenosine monophosphate,cyclic adenosine,cyclic triadenylate and cyclic tetraadenylate.Furthermore,these cyclic adenylate products may be linked in a different way from the common 3’-5’ linkage,which needs further experimental confirmation.Biochemical experiment shows that the three mutants of AtCdnG(D73N/D75N/D132N)are completely inactiveted.The NTase domain protein(1-349 or 1-379)had no cyclase activity and only the full-length protein had cyclase activity,suggesting that the C-terminal domain regulates AtCdnG enzyme activity.This is obviously different from the reported bacterial CDNTases,their NTase domain remains the enzyme activity like full-length protein.Based on the existing experimental results,our main conclusions are as follows:1.Although the structure of AtCdnG is similar to the typical NTase domain,there are also obvious differences near the active center,which may be related to its catalytic specificity;2.The substrates of AtCdnG has a preference for ATP and has a variety of catalytic products,and the connection mode may be 2’-5’ linkage;3.The C-terminal domain of AtCdnG acts as a switch to regulate the cyclase activity,which is the first report of bacterial CD-NTase that requires dual domains for cyclase activity.In conclusion,the results of the study will provide a soiled basis for the elucidation of catalytic mechanism,and will also provide a theoretical basis for the further study of the functional mechanism of bacterial CBASS. |
