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

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.