| Research backgroundInnate immunity is the first line of host defense against invading pathogens.The cGAS-cGAMP-STING mediated signal pathway is an important signal to activate the host's natural immune system.cGAS(cyclic GMP-AMP Synthase)is a double-stranded DNA(dsDNA)sensor in mammalian cells,when mammalian cells are infected by pathogenic microorganisms,cGAS recognizes the dsDNA of pathogenic microorganisms and synthesizes 2'3'-cGAMP.2'3'-cGAMP activates STING(Stimulator of Interferon Genes)protein,activated STING recruits TANK-binding kinase 1(TBK1),which phosphorylates STING and subsequently the transcription factor IFN regulatory factor 3(IRF3).Phosphorylated IRF3 dimerizes and translocated into the nucleus,accelerating expression of type ? IFNs and inflammatory cytokines,leading to antiviral immune responses.2'3'-cGAMP(cyclic GMP-AMP)is a member of the cyclic dinucleotides(CDNs)family.Cyclic dinucleotides are new second messengers,which control various important biological processes,such as biofilm development,motility,and pathogenicity in bacteria,and the innate immune response in mammalian cells.The cGAS-cGAMP-STING pathway mediates the human body's host defense against a variety of DNA viruses.Its similar pathways also exist in bacteria.The messenger molecule 3'3'-cGAMP that belongs to the cyclic dinucleotide,which participates in important way of bacteria to resist phage infection.The cGAS-cGAMP-STING pathway is mediated to provide host defense against a variety of DNA viruses,such as cGAS recognizes the dsDNA of adenovirus or HIV virus,produces type I interferon,and triggers innate immune response.However,many viruses have also evolved corresponding immune escape mechanisms that interfere and disrupt the host's immune response.Such as pathogens targeting cGAS to evade cytosolic DNA sensing,pathogen degradation 2'3'-cGAMP hinders STING activation,etc.KSHV(Kaposi's Sarcoma-associated Herpesvirus)is a human y herpesvirus,which is the causative agent of malignant diseases such as Kaposi's sarcoma(KS)and primary effusion lymphoma.Recently,it was found that the tegument protein ORF52(also named KicGAS)encoded by the open reading frame 52(orf52)gene of KSHV can specifically inhibit the enzyme activity of cGAS,thus escaping the host's innate immunity.Recently,functional cGAS-cGAMP-STING pathway has also been found in bacteria,which plays an important role in the bacteria's resistance to phage infections.For example,when Vibrio cholerae is infected by phages,it somehow triggers DncV(Dinucleotide cyclase in Vibrio)belonging to the cGAS/DncV-like nucleotide transferase super family to synthesize 3'3'-cGAMP,which activates the phospholipase CapV(cGAMP-activated phospholipase in Vibrio),CapV degrades membrane phospholipids,leading to the loss of membrane integrity and cell death before completion of phage reproduction.Further research reveals that this antiphage defense system is present in more than 10%of the sequenced bacterial genome,indicating that the cGAS-cGAMP-STING pathway is of ancient evolutionary roots.Therefore,the study of the related proteins that pathogenic microorganisms such as viruses and bacteria regulate or respond to cGAS-cGAMP signals may help to understand the influence of pathogenic microorganisms on human cGAS-cGAMP-STING mediated natural immunity,and provide an important theoretical basis for drug targets and drug research.Based on frontier research,this paper selects KSHV protein ORF52 and Vibrio cholerae protein CapV,and analyzes its protein structure,function and mechanism,and to clarify the molecular structure basis of their regulation and response to cGAScGAMP signal.(1)KSHV virus inhibits cGAS activity through ORF52,but the mechanism by which ORF52 inhibits cGAS enzyme activity is unclear.(2)CapV is a cyclic dinucleotide 3'3'-cGAMP-dependent phospholipase,which is activated after binding to 3'3'-cGAMP to play a role in hydrolyzing membrane phospholipids.However,CapV has no structural homology with the known cGAMP binding protein.CapV binds to 3'3'-cGAMP molecular structure basis and mechanism is still unclear.This paper discusses the above-mentioned issues in order to understand the structural basis and molecular mechanism of ORF52 and CapV in the host's innate immunity.Research contentPreparation and purification of cGAS-cGAMP componentsIn order to study the structural mechanism of ORF52 and CapV mediated cGAScGAMP-STING signal from the perspective of protein structureo We firstly expressed and purified cGAS homologues,included human cGAS(hcGAS)and mouse cGAS(mcGAS).We explore and establish the microbial-based method to prepare these cyclic nucleotides(CDNs),included c-di-GMP,2'3'-cGAMP,3'3'-cGAMP.1.Purification of cGAS homologous proteinsThe Escherichia coli system was used to express cGAS.The expression vector and strains are His-SUMO expression vector and E.coli strain BL21-CodonPlus(DE3)-RIL respectively.We obtained relatively pure hcGAS and mcGAS proteins after purification.2.The fermentation preparation of CDNsProducing high yield of CDNs by engineering the overexpression of the proper dinucleotide cyclases(DNCs)and other related proteins in Escherichia coli.Increase the initial yield of cyclic dinucleotides by optimizing the type of medium,induction temperature and induction time and other factors.(1)the cells co-expressing tDGCm(Thermotoga maritima Diguanylate Cyclase mutant,residues:82-248)and STINGCTD(the carboxy-terminal domain residues of human STINGH232,residues:149-379)in LB medium with 0.1 mM IPTG at 37? for 20 h were chosen for the final protocol.(2)the cells co-expressing DncVtm(DncVT179R,residues:1-419),GMK(Staphylococcus aureus Guanylate Kinase),and NDK(E.coli nucleoside Diphosphate Kinase)in TB medium with 0.1 mM IPTG at 18? for 20 h were chosen for the final protocol.(3)E.coli strain BL21-CodonPlus(DE3)-RIL expressing SUMO-mcGAS in the optimal modified M9 minimal medium with 0.1 mM IPTG at 37? for 20 h was chosen for the final protocol.The c-di-GMP and 3'3'-cGAMP produced by Escherichia coli are in the bacteria,Crude c-di-GMP and 3'3'-cGAMP are obtained through mini ultra high pressure cell disrupter,centrifugation filtration and other steps.the produced 2'3'-cGAMP is filtered in the culture solution to obtain a crude product of 2'3'-cGAMP.Purifying the bacteriaproduced CDNs by a unified and simple process involving a STING affinity column,macroporous adsorption resin and C1 8 reverse-phase liquid chromatography.(4)No obvious impurities were detected by HPLC with UV detection at the 254 nm wavelength.LC/MS clearly identified the pure products as the target CDNs.We obtained the diammonium salts of c-di-GMP,3'3'-cGAMP and 2'3'-cGAMP with weight purity of>99,>96,>99%and in yields of>68,>26,and>82 milligrams per liter of culture,respectively.The purified c-di-GMP or 3'3'-cGAMP bound to STING had a dissociation constant Kd of 4.09 ?M or 5.40 ?M,and the purified 2'3'-cGAMP to STING is endothermic with a dissociation constant Kd of?16 nM,but at this high affinity,curve fitting may not be precise.The crystal structure of herpesvirus tegument protein ORF52 and its inhibition mechanism of cGASKSHV inhibits cGAS activity through ORF52,but the structural mechanism by which ORF52 inhibits cGAS activity is unclear.In order to study the effect of ORF52 inhibits cGAS-cGAMP-STING pathway from the perspective of protein structure,we firstly purified the protein of ORF52 of different species,and then explored the crystal growth conditions of ORF52,and the regulation of ORF52 inhibits the cGAS.1.Purification of ORF52 homologous proteinsORF52 homologous were also expressed in E.coli system from different sources,included the murine ORF52(Mh52,hereinafter called Mh52)and porcine ORF52(Por52,hereinafter called Por52).After three steps purification,included Ni-NTA affinity chromatography,strong cation exchange chromatography and Size-exclusion chromatography,we obtained high purity Mh52 and Por52 proteins.2.Crystal preparation and structure analysis of Mh52We screened and optimized the crystals of two ORF52 homologous proteins,and obtained a single crystal of Mh52 with resolution of 2.50 A.Through molecular replacement and model modification,the crystal structure of Mh52 was finally obtained.The Rwork and Rfree are 21.08%and 26.88%,respectively,and the Ramachandran plot shows 99.19%residues are in favored zone,and no residues fall into the disallowed zone.The crystal structure of Mh52 contains amino acid residues 46-105,and its Nterminal 1-45 and C-terminal 106-135 are not built due to the invisible density.We used the co-crystal method to screen the Mh52-dsDNA binary complex and Mh52-DNAcGAS ternary complex,but no satisfactory complex crystals were obtained.3.The function of Mh52Pull-down experiments showed that there is a weaker direct interaction between Mh52 protein and cGAS,but the addition of foreign DNA can enhance the interaction.Enzyme activity experiments showed that when the ratio of Mh52 to cGAS concentration is ?20:1,cGAS enzyme activity can be completely inhibited.Mh52 protein can bind to dsDNA,with a high affinity for 45-59 bp double-stranded DNA while weaker for 18-33 bp dsDNA.The binding of dsDNA has no sequence selectivity and is length-dependent.Based on these,we preliminarily speculate that Mh52 protein not only competes with cGAS for binding to dsDNA,but also affects the mode of action of cGAS and DNA through direct interaction,thereby preventing the enzyme activity of cGAS from being effectively activated by dsDNA and inhibiting the enzyme activity of cGAS.The structural basis and function of cGAMP-activated phospholipase CapVThe activation of phospholipase CapV is cGAMP-dependent.CapV belongs to the Patatin-like phospholipase family and is a new cGAMP binding protein.Therefore,we expressed and purified CapV protein from different bacterial sources,and then coincubate with the prepared 3'3'-cGAMP to obtain complex crystals.We determined the structure of abCapV and abCapV-3'3'-cGAMP,confirmed that CapV can directly bind to cGAMP and exert immune response function.1.Purification of CapV full-length proteinUsing the E.coli expression system to express vcCapV derived from Vibrio cholerae and abCapV derived from we constructed vcCapV and abCapV into His-SUMO expression vectors.A large number of soluble proteins were obtained after expression and purification,but the protein properties were not good.vcCapV tend to polymerize,which was not conducive to purification and the search of crystal conditions.By optimizing the purification conditions of abCapV,a large number of stable dimeric full-length abCapV proteins were obtained.2.Purification of CapV mutantsIn order to verify the CapV enzyme activity and Conducive to crystal growth,we expressed and purified mutant proteins of vcCapV and abCapV,and obtained vcCapV active center mutant proteins(S62A,D201A,D201N).We obtained the active center mutant protein of abCapV(S49A,D174A)and the key amino acid mutant protein of the two-dimer interaction(W228A).In order to verify the function of the abCapV,the key amino acid mutant proteins(K133A,E189A,D143A,Y192A,T131A,H258A,L140S,R142G)that interact with 3'3'-cGAMP and C-terminal protein(residues:1-310,His-MBP vector)were purified.3.Crystal preparation and structure analysis of abCapVBy screening and optimizing the crystal conditions of abCapV,the crystal obtained was collected using Shanghai Synchrotron Radiation Facility(SSRF)to obtain highresolution diffraction data with a resolution of 2.30 A.The Rwork and Rfree are 22.14%and 24.43%,respectively,and the Ramachandran plot shows 96.67%residues are in favored zone,and no residues fall into the disallowed zone.Then the three-dimensional structure of abCapV was obtained through molecular replacement,model building,structure modification,etc.The form of abCapV adopt a dimeric structure,which is consistent with the dimer peak and the functional dimer unit used in crystallization.The structure of abCapV protein is similar to phospholipase Patatin-17(RMSD is 1.844 A),abCapV adopts a structure and topology that are typical of ?/? hydrolase with approximately three layers,basically ?/?/? in content,in which a central six-stranded?-sheet is sandwiched essentially between ?-helices front and back.The active site also contains a Ser-Asp catalytic dyad.The main differences between the two proteins lie in three places,including loop1 area(residues:82-92),loop2 area(residues:211-220)and C terminal(residues:320-340).4.The structure of abCapV with 3'3'-cGAMP complexThe crystal of abCapV combined with 3'3'-cGAMP was obtained by the co-crystal method,and the complex single crystal was obtained after crystal optimization.Highresolution diffraction data with a resolution of 2.76 A was collected using Shanghai Synchrotron Radiation Facility(SSRF).Then the three-dimensional structure of abCapV-3'3'-cGAMP complex is obtained,through molecular replacement,model building,structure modification,etc.The Rwork and Rfree are 27.04%and 29.29%,respectively.The three-dimensional structure of abCapV in apo and 3'3'-cGAMP states is basically similar(RMSD is 0.5775 A),the main difference lies in the loop1 region.5.Structure and function analysis of abCapVThrough further structural analysis and functional research,we have obtained the following main results:(1)abCapV is a dimer in the crystal structure and in the solution state;(2)abCapV binds to 3'3'-cGAMP with high affinity(Kd:10.6 nM)and the ratio of 2:2.Its binding region is located in another cavity next to the active site.The two cavities are separated by ?10 and all.The residues involved in the interaction are conserved residues,including Glu189,Lys133,Arg142,Leu140,Asp143,His188,which is a new type of cyclic nucleotide binding;(3)The enzyme active site is also SerAsp catalytic dyad,S49A and D174A mutants are all inactive mutants;(4)The loop1 region plays a key role in the recognition of phospholipid substrates;(5)The molecular mechanism of abCapV catalyzed degradation of phospholipids:the binding of 3'3'cGAMP promotes conformational changes and protein oligomerization in the loopl region of abCapV,thereby abCapV can specifically recognizes and binds the substrate phospholipids,and then use the classic cPLA2(cytosolic Phospholipase A2)phospholipase similar catalytic mechanism to efficiently degrade phospholipids into free fatty acids.Conclusions,Innovations,DeficienciesConclusions1.The low-cost microbial fermentation method can efficiently prepare CDNs(c-diGMP,3'3'-cGAMP,2'3'-cGAMP)2.Mh52 binds to dsDNA with no sequence selectivity but length-dependent.3.Mh52 inhibits cGAS enzyme activity by interacting with cGAS and competing for binding to dsDNA.4.The overall structure of abCapV protein is similar to that of phospholipase Patatin17,basically showing a three-layer ?/?/? pattern.5.The binding region of abCapV and 3'3'-cGAMP is located in another cavity next to the active site.The two cavities are separated by ?10 and ?11,which is a new type of 3'3'-cGAMP binding protein.6.The combination of 3'3'-cGAMP promotes conformational changes and protein oligomerization in the loopl region of abCapV,and then abCapV uses a catalytic mechanism similar to cPLA2 phospholipase to efficiently degrade substrate phospholipids.Innovations1.It is the first report to use microbial fermentation to prepare high-purity CDNs.2.For the first time,it is determined that Mh52 inhibits cGAS activity through competitive binding dsDNA.Mh52 tends to bind 45-59 bp dsDNA with no sequence selectivity.3.The crystal structure of phospholipase abCapV and its complex with 3'3'-cGAMP is reported for the first time,and the mechanism by which 3'3'-cGAMP activates phospholipase abCapV to hydrolyze phospholipids has been determined.Deficiencies1.Because Mh52 protein is easily degraded,we didn't obtain full-length Mh52 protein crystals.The conditions for crystal screening need to be further optimized.This work needs further research.2.Mh52-dsDNA binary complex and Mh52-dsDNA-cGAS ternary complex crystal screening conditions need further study.3.The specific mechanism of hydrolysis of phospholipid by abCapV combined with 3'3'-cGAMP needs to be further studied. |
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