Structural And Functional Studies Of C-di-AMP Phosphodiesterase Rv2837c And C-di-GMP-dependent Transcriptional Factor PA4878

Bacteria can accurately sense and respond to the changes of the surrounding environment such as nutrition,stress,temperature,osmolarity and PH.This ability is critical for the survival of bacteria.Second messengers are crucial in the transduction of signals and cyclic dinucleotides second messengers have over the years been demonstrated to be central to this process.It has been reported that c-di-GMP and c-di-AMP can regulate many bacterial physiological activities,including mobility,toxicity,size,cell wall homeostasis and biofilm formation.Meanwhile,c-di-GMP and c-di-AMP can bind to STING then inducing type 1 interferon response in the process of the infected host.The first part of this paper mainly focused on structural biology and enzyme activity analysis about the Rv2837c from Mycobacterium tuberculosis as a c-di-AMP phosphodiesterase.Gram-positive bacterium M.tuberculosis is an intracellular pathogen that causes tuberculosis,which remains one of the world’s deadliest communicable diseases.Our knowledge about this causative agent is still limited.Therefore,it is essential to understand the biology of this pathogen in order to eradicate the infection and develop therapeutic vaccines.Similar to some another gram-positive pathogens,M.tuberculosis contains c-di-AMP signaling system.CnpB(Rv2837c)as a phosphodiesterase is essential for c-di-AMP homeostasis in M.tuberculosis.When infected bone marrow derived macrophages and mice with M.tuberculosis wild type(WT)and △cnpB strains,the results showed that the AcnpB infected macrophages secreted 10-fold more IFN-β than those infected with the WT and AcnpB in M.tuberculosis attenuates virulence during infection of mice.As early as 2012,Rv2837c was reported as a bifunctional protein with the ability to degrade nanoRNA and to dephosphorylate 3’-phosphoadenosine 5’-phosphate(pAp)to AMP,as the NrnA homolog protein.Rv2837c has degradation activity of various substrates,whether the degradation mechanism of each substrate are identical.The degradation activity for different substrate was different,however,the molecular mechanism for the different remains unknown.Firstly,we reported the crystal structure of Rv2837c(2.0 A),by structure analysis and mutants enzyme activity measure confirmed that Rv2837c hydrolyze its substrate with a two Mn-based mechanism.The multiple sequence alignment also showed that the mechanism of the two metal involved in the degradation reaction was universal.Secondly,we reported Rv2837c with c-di-AMP hydrolysis intermediate 5’-pApA complex structure(2.35 A).Because 5’-pApA is a perfect nanoRNA,the complex structure of Rv2837c/5’-pApA also provides an excellent model of Rv2837c hydrolyzing nanoRNA.Based on the complex structure and biochemical data we propose a simplified catalytic mechanism for phosphodiester bond hydrolyzation by Rv2837c.Thirdly,We tested the PDE activities of Rv2837c toward cyclic dinucleotides(c-di-AMP,c-di-GMP,3’-5’cGAMP)and linear dinucleotides(pGpG,pApA),confirmed that Rv2837c specifically targets 3’-5’ phosphodiester bond,and determined that Rv2837c can degrade 2’-5’cGAMP into 2’-5’pGpA.According to our structures and calculated values strongly suggest that Rv2837c hydrolyze c-di-AMP to pApA at first step,then the intermediate pApA flip in Rv2837c active site to produce AMP.Finally we overexpressed Rv2837c in M.smegmatis of the MSMEG2630 deletion and MG1655 to explore the c-di-NMP phosphodiesterase activity of Rv2837c in vivo.The results showed that Rv2837c may play a role in both c-di-AMP and c-di-GMP signaling in vivo.The second part of this paper mainly focused on structural biology and physiological function analysis about the BrlR from Pseudomonas aeruginosa which is known to be activated by c-di-GMP.P.aeruginosa is an opportunistic Gram-negative bacterium that is primarily responsible for various types of infections,including cystic fibrosis(CF),burn wounds,and urinary tract infections,and HIV-related illness and these infections are difficult to eradicate.P.aeruginosa can cause so many infections mainly depends on its powerful virulence factor system and the difficulty for clinical anti infective therapy primarily because of the bacterial resistance to antibiotics.In 2012,Karin Sauer et.al reported that BrlR as a member of the MerR family can regulate the gene expression which associated with biofilm resistance and self expression only in bacterial biofilm.The following studies show that BrlR binds to the Pbrlr and PmexA,PmexE DNA sequence,then activates the efflux pump expression to enhance bacterial resistance.Unlike known MerR multidrug transport activators,BrlR and brlR expression are not activated by multidrug transporter substrates.Instead,BrlR-DNA binding was enhanced by c-di-GMP.At first,we want to understand c-di-GMP and BrlR binding mechanism by structural analysis,however,with the further studies,we found that BrlR is a very complex transcriptional regulatory factor by multiple signaling pathways.In this study,we have solved crystal structures of apo BrlR(3.1 A)and BrlR in complex with c-di-GMP(2.5 A).Our structures showed that BrlR harbors two independent c-di-GMP binding sites in its DNA-binding domain that bind a stacked c-di-GMP molecule respectively.The residues involved in two c-di-GMPs binding arenot the conserved DNA-binding residues,and the binding mode of BrlR is unlike any c-di-GMP receptors discovered to date.Unlike other MerR family proteins,the relative position of two terminal motifs of BrlR has a unique conformation,in which the HTHmotif of DNA-binding domain was self-blocked by the multidrug-binding domain and may result in the weaker BrlR-DNA binding.Moreover,the C-terminal domain of BrlR adopts a typical fold of the multidrug-binding domain,which adapts for small-molecule binding.We found that the BrlR is able to combine with diverse toxic compounds including the pyocyanin pigment of P.aeruginosa.Our crystal structure of BrlR-C domain bound to an analog of pyocyanin(1.4 A)clearly showed that BrlR contains a rigid binding pocket for pyocyanin,the analog binding in the multidrug-binding domain occupies the interface of two terminal domains and significantly relocates the DNA-binding domain of BrlR,which well explains that pyocyanin contributes to the DNA-binding of BrlR more efficiently.Biochemical data and in vivo data also validate this new feature of BrlR.Overall,these findings indicate that BrlR is a novel dual-receptor which combines with both c-di-GMP and pyocyanin via its two terminal domains respectively,and provides us with new insights into the link between pyocyanin production and antibiotic resistance of P.aeruginosa.