posted on 2021-03-09, 22:03authored byLing Li, Lian He, Bo Wu, Chuandi Yu, Hongxin Zhao, Yubin Zhou, Junfeng Wang, Lei Zhu
OptoPB is an optogenetic tool engineered
by fusion of the phosphoinositide
(PI)-binding polybasic domain of Rit1 (Rit-PB) to a photoreactive
light-oxygen-voltage (LOV) domain. OptoPB selectively and reversibly
binds the plasma membrane (PM) under blue light excitation, and in
the dark, it releases back to the cytoplasm. However, the molecular
mechanism of optical regulation and lipid recognition is still unclear.
Here using nuclear magnetic resonance (NMR) spectroscopy, liposome
pulldown assay, and surface plasmon resonance (SPR), we find that
OptoPB binds to membrane mimetics containing di- or triphosphorylated
phosphatidylinositols, particularly phosphatidylinositol 4,5-bisphosphate
(PI(4,5)P2), an acidic phospholipid predominantly located
in the eukaryotic PM. In the dark, steric hindrance prevented this
protein–membrane interaction, while 470 nm blue light illumination
activated it. NMR titration and site-directed mutagenesis revealed
that both cationic and hydrophobic Rit-PB residues are essential to
the membrane interaction, indicating that OptoPB binds the membrane via a specific PI(4,5)P2-dependent mechanism.