7yib
From Proteopedia
Crystal structure of wild-type Cap4 SAVED domain-containing receptor from Enterobacter cloacae
Structural highlights
FunctionCAP4_ENTCL CBASS (cyclic oligonucleotide-based antiphage signaling system) provides immunity against bacteriophage. The CD-NTase protein synthesizes cyclic nucleotides in response to infection; these serve as specific second messenger signals. The signals activate a diverse range of effectors, leading to bacterial cell death and thus abortive phage infection. A type II-C(AAG) CBASS system (PubMed:32839535).[1] [2] Binds second messenger 3',3',3'-cyclic AMP-AMP-GMP (cAAG). In the presence of 3',3',3'-cyclic AMP-AMP-GMP (synthesized by the cognate CD-NTase protein in the CBASS operon), endonucleolytically degrades dsDNA to approximately 17 bp length fragments, with a preference for 5'-C|NG sites. Only binds DNA in the presence of cAAG. Not activated by c-di-AMP, c-di-GMP, 3'3'-cyclic GMP-AMP (3'3'-cGAMP) or the second messenger of A.baumanii strain ATCC 27244.[3] Protects E.coli against phage T2 infection. When the cdnD-cap2-cap3-cap4 operon is introduced in E.coli there is a more than 10(3) decrease in the efficiency of T2 plaque formation. The operon does not protect against phage T5 and only about 10-fold against T7.[4] Publication Abstract from PubMedUnder selective pressure, bacteria have evolved diverse defense systems against phage infections. The SMODS-associated and fused to various effector domains (SAVED)-domain containing proteins were identified as major downstream effectors in cyclic oligonucleotide-based antiphage signaling system (CBASS) for bacterial defense. Recent study structurally characterizes a cGAS/DncV-like nucleotidyltransferase (CD-NTase)-associated protein 4 from Acinetobacter baumannii (AbCap4) in complex with 2'3'3'-cyclic AMP-AMP-AMP (cAAA). However, the homologue Cap4 from Enterobacter cloacae (EcCap4) is activated by 3'3'3'-cyclic AMP-AMP-GMP (cAAG). To elucidate the ligand specificity of Cap4 proteins, we determined the crystal structures of full-length wild-type and K74A mutant of EcCap4 to 2.18 and 2.42 A resolution, respectively. The DNA endonuclease domain of EcCap4 shares similar catalytic mechanism with type II restriction endonuclease. Mutating the key residue K74 in the conserved DX(n)(D/E)XK motif completely abolishes its DNA degradation activity. The potential ligand-binding cavity of EcCap4 SAVED domain is located adjacent to its N-terminal domain, significantly differing from the centrally located cavity of AbCap4 SAVED domain which recognizes cAAA. Based on structural and bioinformatic analysis, we found that Cap4 proteins can be classified into two types: the type I Cap4, like AbCap4, recognize cAAA and the type II Cap4, like EcCap4, bind cAAG. Several conserved residues identified at the surface of potential ligand-binding pocket of EcCap4 SAVED domain are confirmed by ITC experiment for their direct binding roles for cAAG. Changing Q351, T391 and R392 to alanine abolished the binding of cAAG by EcCap4 and significantly reduced the anti-phage ability of the E. cloacae CBASS system constituting EcCdnD (CD-NTase in clade D) and EcCap4. In summary, we revealed the molecular basis for specific cAAG recognition by the C-terminal SAVED domain of EcCap4 and demonstrates the structural differences for ligand discrimination among different SAVED-domain containing proteins. Specific recognition of cyclic oligonucleotides by Cap4 for phage infection.,Chang JJ, You BJ, Tien N, Wang YC, Yang CS, Hou MH, Chen Y Int J Biol Macromol. 2023 May 15;237:123656. doi: 10.1016/j.ijbiomac.2023.123656. , Epub 2023 Feb 14. PMID:36796558[5] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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