Stabilization of Human Multidrug Resistance Protein 4 (MRP4/ABCC4) Using Novel Solubilization Agents

Abstract

Membrane proteins (MPs) are important drug discovery targets for a wide range of diseases. However, elucidating the structure and function of native MP is notoriously challenging as their original structure has to be maintained once removed from the lipid bilayer. Conventionally, detergents have been used to solubilize MP with varying degrees of success concerning MP stability. To try to address this, new, more stabilizing agents have been developed, such as calixarene-based detergents and styrene–maleic acid (SMA) copolymer. Calixarene-based detergents exhibit enhanced solubilizing and stabilizing properties compared with conventional detergents, whereas SMA is able to extract MPs with their surrounding lipids, forming a nanodisc structure. Here we report a comparative study using classical detergents, calixarene-based detergents, and SMA to assess the solubilization and stabilization of the human ABC transporter MRP4 (multidrug resistance protein 4/ABCC4). We show that both SMA and calixarene-based detergents have a higher solubility efficiency (at least 80%) than conventional detergents, and show striking overstabilization features of MRP4 (up to 70 °C) with at least 30 °C stability improvement in comparison with the best conventional detergents. These solubilizing agents were successfully used to purify aggregate-free, homogenous and stable MRP4, with sevenfold higher yield for C4C7 calixarene detergent in comparison with SMA. This work paves the way to MRP4 structural and functional investigations and illustrates once more the high value of using calixarene-based detergent or SMA as versatile and efficient tools to study MP, and eventually enable drug discovery of challenging and highly druggable targets.

CD81 extracted in SMALP nanodiscs comprises two distinct protein populations within a lipid environment enriched with negatively charged headgroups

“…This contrasts with a previous report where SMA was used to solubilise the ABC transporter MRP4/ABBC4 (multidrug resistance protein 4) from insect cell membranes, where the changes in turbidity were mirrored by protein specific solubilisation [ 42 ]. Secondly the speed of CD81 solubilisation was much slower than that observed for MRP4 [ 42 ], possibly related to the differences in lipid composition of yeast membranes compared to insect cells [ 43 ]. Thus this highlights the importance of measuring protein-specific solubilisation efficiency and that optimal conditions will likely be both protein and expression-system dependent.…”

Extraction and reconstitution of membrane proteins into lipid nanodiscs encased by zwitterionic styrene-maleic amide copolymers

“…One potential advantage of the reconstitution in zSMALPs vs solubilization in detergent is membrane protein stabilization 36,[39][40][41][42] . Figure 5 shows that stabilization does indeed occur following zSMALP formation (see Supplementary Fig.…”

Multivalent and Multifunctional Calixarenes in Bionanotechnology

“…It was proposed and partially proved that these amphiphiles freeze the monomeric structure of the protein, preventing its aggregation and denaturation, through hydrophobic interactions with the protein membrane region and through a network of salt bridges with the basic residues at the cytosol-membrane interface. With the aim of overcoming what has been indicated as a bottleneck in the membrane protein structural biology, [75] in the latest years other studies appeared where the stabilisation, isolation and characterisation of membrane proteins such as Human Multidrug Resistance Protein 4 [76] and G protein-coupled receptors, [77] using similar calixarene detergents, were reported. A glycocalixarene having glucose units at the upper and an heptyl chain at the lower rim also showed to preserve and stabilise, even over long periods of time, the function of three native membrane proteins.…”