Structural Determinants for Light-Dependent Membrane Binding of a Photoswitchable Polybasic Domain
journal contributionposted on 09.03.2021, 22:03 by Ling 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.
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SPRliposome pulldown assaylight illuminationphotoreactive light-oxygen-voltageLight-Dependent Membrane Bindingmembrane mimeticslipid recognitioneukaryotic PMPIacidic phospholipidmembrane interactionlight excitationStructural Determinantstriphosphorylated phosphatidylinositolsRit 1site-directed mutagenesis470 nmoptogenetic toolplasma membranePhotoswitchable Polybasic Domain OptoPBRit-PB residuessurface plasmon resonanceLOVsteric hindrancemechanismNMR titration