Lee Kyu-Yeon, Cheon Seung-Ho, Kim Dong-Gyun, Lee Sang Jae, Lee Bong-Jin
Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 08826, Republic of Korea.
PAL-XFEL, Pohang Accelerator Laboratory, POSTECH, Pohang, Gyeongbuk 37673, Republic of Korea.
IUCrJ. 2020 Apr 17;7(Pt 3):509-521. doi: 10.1107/S2052252520003917. eCollection 2020 May 1.
TatD has been thoroughly investigated as a DNA-repair enzyme and an apoptotic nuclease, and still-unknown TatD-related DNases are considered to play crucial cellular roles. However, studies of TatD from Gram-positive bacteria have been hindered by an absence of atomic detail and the resulting inability to determine function from structure. In this study, an X-ray crystal structure of SAV0491, which is the TatD enzyme from the Gram-positive bacterium (TatD), is reported at a high resolution of 1.85 Å with a detailed atomic description. Although TatD has the common TIM-barrel fold shared by most TatD-related homologs, and PDB entry 2gzx shares 100% sequence identity with SAV0491, the crystal structure of TatD revealed a unique binding mode of two phosphates interacting with two Ni ions. Through a functional study, it was verified that TatD has Mg-dependent nuclease activity as a DNase and an RNase. In addition, structural comparison with TatD homologs and the identification of key residues contributing to the binding mode of Ni ions and phosphates allowed mutational studies to be performed that revealed the catalytic mechanism of TatD. Among the key residues composing the active site, the acidic residues Glu92 and Glu202 had a critical impact on catalysis by TatD. Furthermore, based on the binding mode of the two phosphates and structural insights, a putative DNA-binding mode of TatD was proposed using docking. Overall, these findings may serve as a good basis for understanding the relationship between the structure and function of TatD proteins from Gram-positive bacteria and may provide critical insights into the DNA-binding mode of TatD.
TatD作为一种DNA修复酶和凋亡核酸酶已被深入研究，人们认为仍未知的与TatD相关的脱氧核糖核酸酶在细胞中发挥着关键作用。然而，由于缺乏原子细节以及由此导致的无法从结构确定功能，革兰氏阳性菌中TatD的研究受到了阻碍。在本研究中，报道了革兰氏阳性菌（TatD）中的TatD酶SAV0491的X射线晶体结构，分辨率高达1.85 Å，并对原子进行了详细描述。尽管TatD具有大多数与TatD相关的同源物共有的常见TIM桶状折叠，且蛋白质数据银行（PDB）条目2gzx与SAV0491的序列同一性为100%，但TatD的晶体结构揭示了两个磷酸根与两个镍离子相互作用的独特结合模式。通过功能研究，证实了TatD作为脱氧核糖核酸酶和核糖核酸酶具有镁依赖性核酸酶活性。此外，与TatD同源物的结构比较以及对镍离子和磷酸根结合模式起关键作用的残基的鉴定，使得能够进行突变研究，从而揭示了TatD的催化机制。在构成活性位点的关键残基中，酸性残基Glu92和Glu202对TatD的催化作用有至关重要的影响。此外，基于两个磷酸根的结合模式和结构见解，利用对接提出了TatD假定的DNA结合模式。总体而言，这些发现可能为理解革兰氏阳性菌中TatD蛋白的结构与功能之间的关系提供良好基础，并可能为TatD的DNA结合模式提供关键见解。
