Structural Biology, Max-Delbrück-Center for Molecular Medicine in the Helmholtz Association (MDC), 13125 Berlin, Germany.
Biochemical Institute, Kiel University, Kiel, Germany.
Nucleic Acids Res. 2024 Jan 11;52(1):370-384. doi: 10.1093/nar/gkad1114.
The phospholipase D (PLD) family is comprised of enzymes bearing phospholipase activity towards lipids or endo- and exonuclease activity towards nucleic acids. PLD3 is synthesized as a type II transmembrane protein and proteolytically cleaved in lysosomes, yielding a soluble active form. The deficiency of PLD3 leads to the slowed degradation of nucleic acids in lysosomes and chronic activation of nucleic acid-specific intracellular toll-like receptors. While the mechanism of PLD phospholipase activity has been extensively characterized, not much is known about how PLDs bind and hydrolyze nucleic acids. Here, we determined the high-resolution crystal structure of the luminal N-glycosylated domain of human PLD3 in its apo- and single-stranded DNA-bound forms. PLD3 has a typical phospholipase fold and forms homodimers with two independent catalytic centers via a newly identified dimerization interface. The structure of PLD3 in complex with an ssDNA-derived thymidine product in the catalytic center provides insights into the substrate binding mode of nucleic acids in the PLD family. Our structural data suggest a mechanism for substrate binding and nuclease activity in the PLD family and provide the structural basis to design immunomodulatory drugs targeting PLD3.
磷脂酶 D（PLD）家族由具有针对脂质的磷脂酶活性或针对核酸的内切核酸酶和外切核酸酶活性的酶组成。PLD3 作为 II 型跨膜蛋白合成，并在溶酶体中经蛋白水解切割，产生可溶性活性形式。PLD3 的缺乏导致溶酶体中核酸的降解速度减慢，并慢性激活核酸特异性细胞内 Toll 样受体。虽然 PLD 磷脂酶活性的机制已得到广泛研究，但对于 PLD 如何结合和水解核酸知之甚少。在这里，我们确定了人 PLD3 的无配体和单链 DNA 结合形式的腔 N-糖基化结构域的高分辨率晶体结构。PLD3 具有典型的磷脂酶折叠，并通过新鉴定的二聚化界面形成两个独立催化中心的同源二聚体。在催化中心与来自 ssDNA 的胸苷产物的复合物中的 PLD3 结构提供了关于 PLD 家族中核酸底物结合模式的见解。我们的结构数据表明了 PLD 家族中底物结合和核酸酶活性的机制，并为设计针对 PLD3 的免疫调节药物提供了结构基础。
