Structure and mechanism of the human CTDNEP1-NEP1R1 membrane protein phosphatase complex necessary to maintain ER membrane morphology.
Structure and mechanism of the human CTDNEP1-NEP1R1 membrane protein phosphatase complex necessary to maintain ER membrane morphology.
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维持内质网膜形态所必需的人 CTDNEP1-NEP1R1 膜蛋白磷酸酶复合物的结构和机制。
DOI：
10.1101/2023.11.20.567952
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Airola,MichaelV
中科院分区：
文献类型：
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作者：
Gao,Shujuan;CarrasquilloRodríguez,JakeW;Bahmanyar,Shirin;Airola,MichaelV
C-末端结构域核膜磷酸酶1（CTDNEP 1）是一种非经典蛋白丝氨酸/苏氨酸磷酸酶，其在调节ER膜生物发生中具有保守作用。CTDNEP 1的失活突变与髓母细胞瘤（一种侵袭性儿童癌症）的发生相关。跨膜蛋白核膜磷酸酶1调节亚基1（NEP 1 R1）结合CTDNEP 1，但NEP 1 R1调节CTDNEP 1功能的分子细节尚不清楚。在这里，我们发现，敲低NEP 1 R1产生相同的表型报告的CTDNEP 1在哺乳动物细胞中的损失，建立CTDNEP 1-NEP 1 R1作为一个进化保守的膜蛋白磷酸酶复合物，限制ER的扩张。从机制上讲，NEP 1 R1作为一个激活调节亚基，直接结合并增加CTDNEP 1的磷酸酶活性。通过定义一个最小的NEP 1 R1域足以激活CTDNEP 1，我们确定高分辨率的晶体结构的CTDNEP 1-NEP 1 R1复合物结合到一个肽序列作为假底物。在结构上，NEP 1 R1在远离活性位点的位点与CTDNEP 1接合，以稳定和变构激活CTDNEP 1。底物识别由CTDNEP 1中的保守Arg残基促进，其结合并定向活性位点中的底物肽。总之，这揭示了NEP 1 R1如何调节CTDNEP 1的机制，并解释了癌症相关突变如何抑制CTDNEP 1。
C-terminal Domain Nuclear Envelope Phosphatase 1 (CTDNEP1) is a noncanonical protein serine/threonine phosphatase that has a conserved role in regulating ER membrane biogenesis. Inactivating mutations in CTDNEP1 correlate with the development of medulloblastoma, an aggressive childhood cancer. The transmembrane protein Nuclear Envelope Phosphatase 1 Regulatory Subunit 1 (NEP1R1) binds CTDNEP1, but the molecular details by which NEP1R1 regulates CTDNEP1 function are unclear. Here, we find that knockdown of NEP1R1 generates identical phenotypes to reported loss of CTDNEP1 in mammalian cells, establishing CTDNEP1–NEP1R1 as an evolutionarily conserved membrane protein phosphatase complex that restricts ER expansion. Mechanistically, NEP1R1 acts as an activating regulatory subunit that directly binds and increases the phosphatase activity of CTDNEP1. By defining a minimal NEP1R1 domain sufficient to activate CTDNEP1, we determine high-resolution crystal structures of the CTDNEP1–NEP1R1 complex bound to a peptide sequence acting as a pseudosubstrate. Structurally, NEP1R1 engages CTDNEP1 at a site distant from the active site to stabilize and allosterically activate CTDNEP1. Substrate recognition is facilitated by a conserved Arg residue in CTDNEP1 that binds and orients the substrate peptide in the active site. Together, this reveals mechanisms for how NEP1R1 regulates CTDNEP1 and explains how cancer-associated mutations inactivate CTDNEP1.