Mizutani Natsuki, Yonezawa Yasushige, Nakagawa Atsushi, Okamura Yasushi
Laboratory of Integrative Physiology, Department of Physiology, Graduate School of Medicine, The University of Osaka, Suita, Osaka 565-0871, Japan.
Institute for Protein Research, The University of Osaka, Suita, Osaka 565-0871, Japan.
Proc Natl Acad Sci U S A. 2025 Aug 5;122(31):e2500651122. doi: 10.1073/pnas.2500651122. Epub 2025 Jul 29.
Voltage-sensing phosphatase (VSP) comprises a voltage sensor domain (VSD) and a cytoplasmic catalytic region (CCR), achieving a unique electrochemical signal conversion. Previous studies suggest that phosphatidylinositol 4,5-bisphosphate (PI(4,5)P), a membrane phospholipid known to be critical for activities of diverse voltage-gated ion channels, associates with a linker connecting the VSD with the CCR of VSP and regulates VSD-CCR coupling. However, the details of PI(4,5)P interaction with the linker of VSP remain elusive. Here, we exploit advantage of sensitivity of a fluorescent unnatural amino acid, 3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (Anap), to changes in local environment to study interaction between PI(4,5)P and the linker of VSP (Ci-VSP). We found that a conserved tyrosine residue (Y255) as well as neighboring basic residues interacts with PI(4,5)P and this interaction was maintained in G365A Ci-VSP mutant which lacks the substrate PI(4,5)P at the active site and Ci-VSP/human phosphatase and tensin homolog (PTEN) chimera which does not dephosphorylate PI(4,5)P, indicating that the linker interacts with nonsubstrate, regulatory PI(4,5)P outside the active site. Molecular dynamics simulations demonstrated that the linker formed stable interaction with PI(4,5)P in the activated state. These findings indicate that regulation of coupling to an effector region downstream of the VSD through PI(4,5)P binding to the linker is shared among voltage-dependent membrane proteins.
电压感应磷酸酶（VSP）由一个电压感应结构域（VSD）和一个胞质催化区域（CCR）组成，实现独特的电化学信号转换。先前的研究表明，磷脂酰肌醇4,5-二磷酸（PI(4,5)P）是一种已知对多种电压门控离子通道活性至关重要的膜磷脂，它与连接VSD和VSP的CCR的接头结合，并调节VSD-CCR偶联。然而，PI(4,5)P与VSP接头相互作用的细节仍不清楚。在这里，我们利用荧光非天然氨基酸3-(6-乙酰萘-2-基氨基)-2-氨基丙酸（Anap）对局部环境变化的敏感性来研究PI(4,5)P与VSP（Ci-VSP）接头之间的相互作用。我们发现一个保守的酪氨酸残基（Y255）以及相邻的碱性残基与PI(4,5)P相互作用，并且这种相互作用在活性位点缺乏底物PI(4,5)P的G365A Ci-VSP突变体和不使PI(4,5)P去磷酸化的Ci-VSP/人磷酸酶和张力蛋白同源物（PTEN）嵌合体中得以维持，这表明接头与活性位点外的非底物调节性PI(4,5)P相互作用。分子动力学模拟表明，接头在激活状态下与PI(4,5)P形成稳定的相互作用。这些发现表明，通过PI(4,5)P与接头结合来调节与VSD下游效应区域的偶联在电压依赖性膜蛋白中是共有的。
