Graduate Institute of Integrated Medicine, China Medical University, Taichung 406, Taiwan; Department of Medical Research, China Medical University Hospital, Taichung 406, Taiwan.
Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, China Medical University, Taichung 406, Taiwan.
Int J Biol Macromol. 2023 May 15;237:123656. doi: 10.1016/j.ijbiomac.2023.123656. Epub 2023 Feb 14.
Under 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 Å 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(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.
在选择压力下，细菌已经进化出多种防御系统来对抗噬菌体感染。SMODS 相关和融合到各种效应结构域（SAVED）的蛋白被鉴定为细菌防御中环核苷酸基抗噬菌体信号系统（CBASS）的主要下游效应物。最近的研究结构特征化了一种来自鲍曼不动杆菌（Acinetobacter baumannii）的 cGAS/DncV 样核苷酸转移酶（CD-NTase）相关蛋白 4（AbCap4）与 2'3'3'-环 AMP-AMP-AMP（cAAA）的复合物。然而，来自阴沟肠杆菌（Enterobacter cloacae）的同源物 Cap4（EcCap4）被 3'3'3'-环 AMP-AMP-GMP（cAAG）激活。为了阐明 Cap4 蛋白的配体特异性，我们测定了全长野生型和 K74A 突变型 EcCap4 的晶体结构，分辨率分别为 2.18 和 2.42Å。EcCap4 的 DNA 内切酶结构域与 II 型限制内切酶具有相似的催化机制。突变保守 DX(D/E)XK 基序中的关键残基 K74 完全消除了其 DNA 降解活性。EcCap4 SAVED 结构域的潜在配体结合腔位于其 N 端结构域附近，与位于中央的 AbCap4 SAVED 结构域的腔显著不同，后者识别 cAAA。基于结构和生物信息学分析，我们发现 Cap4 蛋白可以分为两种类型：I 型 Cap4，如 AbCap4，识别 cAAA；而 II 型 Cap4，如 EcCap4，结合 cAAG。通过 ITC 实验验证了 EcCap4 SAVED 结构域潜在配体结合口袋表面鉴定的几个保守残基在直接结合 cAAG 方面的作用。将 Q351、T391 和 R392 突变为丙氨酸会使 EcCap4 无法结合 cAAG，并显著降低由 EcCdnD（D 类中的 CD-NTase）和 EcCap4 构成的阴沟肠杆菌 CBASS 系统的抗噬菌体能力。总之，我们揭示了 EcCap4 C 端 SAVED 结构域特异性识别 cAAG 的分子基础，并证明了不同 SAVED 结构域蛋白之间配体识别的结构差异。
