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

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.

 

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This study describes the use of styrene maleic acid (SMA) copolymer to solubilize and purify the SMO transmembrane domain in a lipid-based membrane mimic, the so-called polymer nanodiscs. The SMA-nanodiscs are shown to be homogeneous and maintain the physiological activity of SMO protein, making it possible to measure the dissociation constant (Kd) for SMO ligands using ligand-based solution nuclear magnetic resonance spectroscopy.

The ability to characterize membrane proteins such as SMO in their native state is important for the development of effective pharmaceutical drugs. By using a lipid-based membrane mimic, the researchers were able to retain the structure and function of SMO protein to the highest extent possible. This work paves the way for investigating the structure, function, and drug development of SMO proteins in a native-like lipid environment, which could have important implications for the treatment of various cancers, including medulloblastoma and basal cell carcinoma.