Structural Determinants for Light-Dependent Membrane Binding of a Photoswitchable Polybasic Domain

Abstract

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)P 2 ), 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)P 2 -dependent mechanism.

Application and Method of Surface Plasmon Resonance Technology in the Preparation and Characterization of Biomedical Nanoparticle Materials

“…This not only confirmed the photosensitivity of OptoPB but also, through mutational analysis of different residues of OptoPB, further revealed the molecular mechanism of its binding to the plasma membrane. 53 Two highly specific techniques were employed, namely sortase tagging and click chemistry, to graft AGuIX (activation and guidance of irradiation X) nanoparticles with two different targeting VHHs (Programmed Death-Ligand 1 (PD-L1, A12 VHH) and Cluster of Differentiation 47 (CD47, A4 VHH)), aiming for combined targeted immunotherapy. During this process, the efficacy of the two conjugation techniques was assessed through SPR experiments.…”

Optophysiology: Illuminating cell physiology with optogenetics

“…To meet the need for real-time control over protein-lipid interactions in living cells, OptoPB was developed by fusing LOV2 with phospholipid-binding polybasic (PB) domains derived from stromal interaction molecules (STIMs) or small GTPases ( 160 , 161 ). The endoplasmic reticulum (ER)-anchored version of OptoPB [termed OptoPBer ( 160 ) or LiMETER (for light-inducible membrane-tethered peripheral ER)] further enabled light-inducible ER-PM junction formation ( 162 ), with intermembrane space subjected to phototuning at nanoscale.…”

Experimental manipulation of phosphoinositide lipids: from cells to organisms

“…However, a system dubbed optoPB, based on a blue-light-sensitive LOV domain enables inducible translocation to the plasma membrane with a single engineered protein [74]. In optoPB, blue light induces a conformational change that exposes a polybasic tail [74,79]. The polybasic tail in turn interacts with the negatively charged plasma membrane, thereby triggering cytosol-to-plasma membrane relocalization using a single chimeric construct (Figure 5C).…”