Structural and mechanistic insights into disease-associated endolysosomal exonucleases PLD3 and PLD4.
Structural and mechanistic insights into disease-associated endolysosomal exonucleases PLD3 and PLD4.
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对疾病相关内溶酶体核酸外切酶 PLD3 和 PLD4 的结构和机制的见解。
DOI：
10.1101/2023.11.20.567917
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发表时间：
2023
期刊：
影响因子：
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通讯作者：
Nemazee,David
中科院分区：
文献类型：
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作者：
Yuan,Meng;Peng,Linghang;Huang,Deli;Gavin,Amanda;Luan,Fangkun;Tran,Jenny;Feng,Ziqi;Zhu,Xueyong;Matteson,Jeanne;Wilson,IanA;Nemazee,David
内溶酶体外切酶PLD3和PLD4（磷脂酶D3和D4）与自身炎症和自身免疫性疾病有关。我们报告了这些酶的结构，以及它们催化的分子基础。该结构显示链内二聚体拓扑结构，在界面处形成基本活性位点。与其他PLD超家族成员一样，PLD3和PLD4携带HxKxxxxD/E基序并参与磷酸二酯键的切割。这些酶以5 ‘ -3 ’的方式消化ssDNA和ssRNA，并被5 ' -磷酸化阻断。我们捕获了载脂蛋白，中间产物和产物状态的结构，并揭示了“连接-释放”两步催化。我们还意外地通过共价3-磷酸组氨酸中间体证明了磷酸酶的活性。PLD4含有一个额外的疏水钳，可以稳定底物，并可能影响寡核苷酸底物偏好和产物释放。PLD3/4疾病相关突变体的生化和结构分析表明，酶活性或热稳定性降低，这可能是疾病相关的基础。此外，这些发现为治疗设计提供了见解。
Endolysosomal exonucleases PLD3 and PLD4 (phospholipases D3 and D4) are associated with autoinflammatory and autoimmune diseases. We report structures of these enzymes, and the molecular basis of their catalysis. The structures reveal an intra-chain dimer topology forming a basic active site at the interface. Like other PLD superfamily members, PLD3 and PLD4 carry HxKxxxxD/E motifs and participate in phosphodiester-bond cleavage. The enzymes digest ssDNA and ssRNA in a 5′-to-3′ manner and are blocked by 5′-phosphorylation. We captured structures in apo, intermediate, and product states and revealed a "link-and-release" two-step catalysis. We also unexpectedly demonstrated phosphatase activity via a covalent 3-phosphohistidine intermediate. PLD4 contains an extra hydrophobic clamp that stabilizes substrate and could affect oligonucleotide substrate preference and product release. Biochemical and structural analysis of disease-associated mutants of PLD3/4 demonstrated reduced enzyme activity or thermostability and the possible basis for disease association. Furthermore, these findings provide insight into therapeutic design.