0428同步年報-2021-全

Life Science 047 Fig. 1 : Overall structure of Ec CdnD. The protein is presented as a ribbon diagram, in which the N and C-terminal α-helices are colored blue and red. The β-strands are green. On the left is the N-terminal NTase domain that contains three essential residues Asp69, Asp71 and Asp121 for catalysis; their side chains are shown as stick models. On the right is the C-terminal helical domain. The first two helices α1 and α2 correspond to the “spine” in other NTases. [Reproduced from Ref. 5] Insights into the Catalytic Path and Regulation of a Bacterial CTN Synthase Mammalian cyclic GMP-AMP synthase (cGAS) and its bacterial homologues produce various cyclic dinucleotides and trinucleotides (CDN & CTN) as second messengers that participate in the defence against viral infections. Structural comparison of EcCdnD, a cGAS-like enzyme from Enterobacter cloacae, with DNA/RNA polymerase indicates plausible catalytic paths that produce CTN. The bound ATP in a reversed orientation might play a role in activity regulation. C yclic nucleotides in organisms from mammals to bacteria serve as second messengers that regulate cellular activities in response to environmental variations. For example, through a phosphorylation cascade triggered by adrenaline, 3',5'-cyclic AMP (cAMP) promotes the breakdown of glycogen in human beings. Mammalian cGAS synthesizes 2',3'-cyclic GMP-AMP (2',3'-cGAMP) upon activation by double-stranded DNA, of which the presence in cytosolic indicates a foreign invasion or mitochondria breakdown. 1 Innate immune responses are then initiated by cGAMP binding to STING (for STimulator of INterferon Genes). Similarly, 3',3'-cGAMP produced by dinucleotide cyclase in Vibrio cholerae (DncV) activates a phospholipase that degrades the bacterial cell membrane. 2 Such suicide mechanisms, also known as CBASS (Cyclic oligonucleotide- Based Anti-phage Signaling Systems), can prevent further spreading of the virus and protect the bacterial population from being destroyed. 3 Recently, scores of newly discovered cGAS/DncV-like nucleotidyltransferases (CD-NTases) were shown to produce a variety of cyclic dinucleotides and trinucleotides (CDN & CTN). 4 These enzymes share a common architecture that comprises an N-terminal core domain similar to the DNA/RNA polymerases and a C-terminal helical domain that shows a greater difference. Instead of base pairing, the substrate specificity of CD-NTase is determined by interactions with protein side chains; the cage-like active- site pocket might allow retention and reorientation of the reaction intermediates. CD-NTase signaling paths are classified into Types I and II, depending on a requirement of an activation step. 3 So far, no CTN- synthesizing Type-II enzyme had been known 3D-structure. In this work, the research team led by Yeh Chen (China Medical University) determined the crystal structure of Ec CdnD in apo and substrate complex forms with multi-wavelength anomalous dispersion and molecular replacement using beamlines TPS 05A and TLS 15A1 of the NSRRC. 5 The apo -form Ec CdnD is folded into two domains like other CD-NTases ( Fig. 1 ). The two N-terminal helices of Type-I enzymes constitute the “spine”, which is not broken until binding to an activator such as cytosolic DNA. In contrast, Type-II enzymes such as Ec CdnD are active by themselves as reflected in a distinct division of the two helices. Structure comparison with Pa CdnD, a CTN-synthesizing Type-I enzyme of the same class, shows more than 3Å Cα deviation and a 50-residue insertion between strands βE and βF that contains helix α4. Ec CdnD synthesizes cyclic 3’-AMP-AMP-GMP (cAAG) but does not bind to GTP in the absence of ATP. The enzyme has two binding sites for ATP, as shown by measurements of isothermal titration calorimetry (ITC). 6 Co-crystallization and soaking experiments with ATP, GTP and their analogues yielded only complex structures with ATP or its analogues ( Fig. 2 , see next page). In a shared mechanism for NTases, ribose O3' (or O2') atom of the acceptor substrate is bound to one metal ion (termed metal A) and attacks the 5'-phosphate of the donor substrate, of which the triphosphate associates with another metal ion (termed metal B; Fig. 3 , see next page). Both metal ions were observed in the crystal structures, associated with the side chains of Asp69, Asp71 and Asp121, the triphosphate of the donor ATP, and coordinating water molecules. The nucleotide occupying the acceptor substrate binding site was, however, in a