Nanodisc single-molecule pulldown to study lipid-protein interactions.

Abstract

Beyond serving structural roles in the cell membrane, many phospholipids, including phosphatidylinositol phosphates (PIPs), are key signaling molecules that regulate a myriad of cellular processes. Specific interactions with PIPs are crucial for the functions of many signaling proteins, highlighting the need for a convenient and robust method to study lipid-protein interactions. Previously, we established a fluorescence microscopy-based lipid single-molecule pulldown (lipid-SiMPull) assay for detecting interactions between fluorescently tagged proteins of interest in whole-cell lysates and small unilamellar vesicles containing phospholipids of interest. Despite unique advantages of the lipid-SiMPull assay, small unilamellar vesicle is not an optimal membrane model due to its instability, heterogeneity in size, and a membrane curvature inconsistent with the relative flatness of the cell membrane. Here, we report the use of lipid Nanodiscs in lipid-SiMPull. Using PIP-protein pairs of known interactions, we show that Nanodiscs containing various PIPs can pull down protein targets specifically, with an estimated detection threshold of K_d in the 10-20 μM range. Remarkably, we find that each Nanodisc is bound by one copy of the protein (or protein dimer), conferring true single-molecule resolution to the assay. Transient interactions are characterized by the rebinding of proteins to individual Nanodiscs, and dissociation rates (k_off) are determined from dwell time analysis. We apply this assay to interrogate structural requirements for the stability of AKT binding of PI(3,4,5)P₃ and find that an intramolecular interaction between the PH domain and kinase domain is critical for stabilizing the AKT-PI(3,4,5)P₃ interaction. This work estalishes the Nanodisc SiMPull assay as a simple and powerful approach for investigating protein-lipid interactions with single-molecule resolution.