Nonsubstrate PI(4,5)P₂ interacts with the interdomain linker to control electrochemical coupling in voltage-sensing phosphatase (VSP).

Abstract

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)P₂), 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)P₂ 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)P₂ and the linker of <i>Ciona intestinalis</i> VSP (Ci-VSP). We found that a conserved tyrosine residue (Y255) as well as neighboring basic residues interacts with PI(4,5)P₂ and this interaction was maintained in G365A Ci-VSP mutant which lacks the substrate PI(4,5)P₂ at the active site and Ci-VSP/human phosphatase and tensin homolog (PTEN) chimera which does not dephosphorylate PI(4,5)P₂, indicating that the linker interacts with nonsubstrate, regulatory PI(4,5)P₂ outside the active site. Molecular dynamics simulations demonstrated that the linker formed stable interaction with PI(4,5)P₂ in the activated state. These findings indicate that regulation of coupling to an effector region downstream of the VSD through PI(4,5)P₂ binding to the linker is shared among voltage-dependent membrane proteins.