Structural And Functional Studies On Porcine Innate Immune Protein STING And Screening Of CDNs Enhancing Immunity

Cyclic-di-guanosine monophosphate(c-di-GMP),c-di-AMP and cGAMP are the second messengers in the ubiquitous bacteria.Cyclic GMP-AMP synthase(cGAS)was identified as a cytoplasmic DNA sensor that activated the type? IFN pathway by synthesizing the second messenger cGAMP.cGAS was shown to be a member of the nucleotidyltransferase family and to be capable of generating cGAMP in vitro from GTP and ATP in the presence of dsDNA.Accumulation of cytosolic DNA activates a host response,STING-mediated activation of the TBK1-IRF3 axis to induce antiviral gene expression.The cyclic dinucleotide(CDN)-stimulator of interferon genes(STING)pathway plays an important role in the detection of viral and bacterial pathogens in animals.Previous studies have shown that the metazoan second messenger cyclic GMP(2',5')-AMP(3',5')(2',3' cGAMP)generated by cyclic GAM-AMP synthase(cGAS)binds STING with high affinity compared with bacterial CDNs such as c-di-GMP,c-diAMP,and 3',3'cGAMP.Despite recent progress indicating that the CDN-binding domain(CBD)of dimeric STING binds asymmetric 2',3' cGAMP preferentially over symmetric 3',3' CDNs,it remains an open question whether STING molecules,such as human STING,adopt a symmetric dimeric conformation to efficiently engage its asymmetric ligand.The nucleotide ring DncV(VC0179)was removed from a LOOP ring,named VC0179-ALOOP,to achieve the activity soluble expression of VC0179-?LOOP.The modified VC0179-?LOOP has greatly improved the enzyme activity and product generation efficiency.Increasing the utilization rate of GTP and ATP and reducing the loss of enzymes greatly reduced the production cost of small molecules.In this study,mouse mcGAS protein was used as an enzyme to catalyze the synthesis of small 2',3'cGAMP molecules,and the successful preparation and purification of a high purity 2',3'cGAMP molecule could satisfy the requirement of the protein crystal to the ligand.Biochemical experiments show that 2 ',3'cGAMP is the endogenous ligand with the highest binding affinity to porcine STING,which is consistent with the affinity of endogenous ligands to the body itself.2',3'cGAMP activates PAM cells to produce IRF3 and IFN-?.Here,structural studies of the CBD from porcine STING(STINGCBD)in complex with CDNs at 1.76-2.6 A resolutions revealed that porcine STINGCBD,unlike its human and mouse counterparts,can adopt an asymmetric ligand-binding pocket to accommodate the CDNs.We observed that the extensive interactions and shape complementarity between asymmetric 2',3' cGAMP and the ligand-binding pocket make it the most preferred ligand for porcine STING and that geometry constraints limit the binding between symmetric 3',3'CDN and porcine STING.The ligand-discrimination mechanism of porcine STING observed here expands our understanding of how the CDN-STING pathway is activated and of its role in antiviral defense.The pathogenic mycobacterium Mycobacterium tuberculosis encodes two members of the DtxR family of metalloregulators,IdeR and MntR.IdeR represses gene expression in response to ferrous iron,while MntR(Rv2788)functions as a manganese-dependent transcriptional repressor,which represses the expression of manganese transporter genes to maintain manganese homeostasis.Although the structural study towards IdeR is in-depth,there is no Mycobacterium tuberculosis MntR(Mt_MntR)structure available.Herein,we report both apo and manganese bound forms of MntR structures from M.tuberculosis.MntR has evolved into two metal ion binding sites like other DtxR proteins and for the first time,we captured the two sites full of metal ion with one Mn2+ ion at the first site and two Mn2+ions at the second binding site(binuclear manganese cluster).The conformation change of MntR resulting from manganese binding could prime the MntR for DNA binding,which is a conserved activation mechanism among DtxR family.