Solubilization, purification, and ligand binding characterization of G protein-coupled receptor SMO in native membrane bilayer using styrene maleic acid copolymer

Abstract

Smoothened (SMO) protein is a member of the G protein-coupled receptor (GPCR) family that is involved in the Hedgehog (Hh) signaling pathway. It is a putative target for treating various cancers, including medulloblastoma and basal cell carcinoma (BCC). Characterizing membrane proteins such as SMO in their native state is highly beneficial for the development of effective pharmaceutical drugs, as their structures and functions are retained to the highest extent in this state. Therefore, although SMO protein is conventionally solubilized in detergent micelles, incorporating the protein in a lipid-based membrane mimic is still required. In this study, we used styrene maleic acid (SMA) copolymer that directly extracted membrane protein and surrounding lipids as well as formed the so-called polymer nanodiscs, to solubilize and purify the SMO transmembrane domain encapsulated by SMA-nanodiscs. The obtained SMA-nanodiscs showed high homogeneity and maintained the physiological activity of SMO protein, thereby enabling the measurement of the dissociation constant (Kd) for SMO ligands SMO-ligands Shh Signaling Antagonist V (SANT-1) and Smoothened Agonist (SAG) using ligand-based solution nuclear magnetic resonance spectroscopy. This work paves the way for investigating the structure, function, and drug development of SMO proteins in a native-like lipid environment.

Chemically‐Induced Lipoprotein Breakdown for Improved Extracellular Vesicle Purification

“…Many studies have reported that nanodiscs can be formed by exposing cell membranes or liposomes to SMA. [25][26][27][29][30][31][32][74][75][76] In the case of EVs, it is possible that increased membrane curvature combined with the glycocalyx makes them less susceptible to SMAinduced nanodisc formation than cell membranes or liposomes. Additionally, differences in the lipid composition and packaging between EVs and cells may also impact SMA membrane accessibility.…”

“…[25][26][27] SMA is primarily used to extract and study membrane proteins in their natural confirmation through solubilization as nanodiscs. [29][30][31][32] Although the copolymer has a broad solubilization capability, it exhibits varying degrees of effectiveness depending on membrane properties. [27,[33][34][35] For example, phospholipid bilayers containing more proteins pose a greater challenge for solubilization.…”

Detergent-Free Isolation of Membrane Proteins and Strategies to Study Them in a Near-Native Membrane Environment

“…ABCG2, one of the ABC binding cassette proteins, has been directly isolated in its physiologically relevant oligomeric state (dimer) and used to investigate protein–drug interactions [ 145 ]. Smoothened (SMO) GPCR protein isolated in SMA-nanodiscs was active and enabled to measure dissociation constant (K d ) for SMO ligands using NMR experiments [ 146 ]. Drosophila nicotinic acetylcholine receptor subunits were isolated into SMA-nanodiscs, and their native interactions with α-bungarotoxin (α-Btx; insecticidal peptide toxin) were studied [ 147 ].…”