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

Abstract

Tetraspanins exert a wide range of cellular functions of broad medical importance. Despite this, their biophysical characteristics are incompletely understood. Only two high-resolution structures of full-length tetraspanins have been solved. One is that of human CD81, which is involved in the infectivity of human pathogens including influenza, HIV, the malarial Plasmodium parasite and hepatitis C virus (HCV). The CD81 crystal structure identifies a cholesterol-binding pocket, which has been suggested to be important in the regulation of tetraspanin function. Here we investigate the use of styrene-maleic anhydride co-polymers (SMA) for the solubilisation and purification of CD81 within a lipid environment. When CD81 was expressed in the yeast Pichia pastoris , it could be solubilised and purified using SMA2000. This SMALP-encapsulated CD81 retained its native folded structure, as determined by the binding of two conformation-sensitive anti-CD81 antibodies. Analysis by size exclusion chromatography revealed two distinct populations of CD81, only one of which bound the HCV glycoprotein, E2. Optimization of expression and buffer conditions increased the proportion of E2-binding competent CD81 protein. Mass spectrometry analysis indicated that the lipid environment surrounding CD81 is enriched with negatively charged lipids. These results establish a platform to study the influence of protein-lipid interactions in tetraspanin biology.

Membrane protein extraction and purification using partially-esterified SMA polymers

“…MRP4/ABCC4 (multidrug resistance protein 4/ATP Binding Cassette transporter C subfamily member 4) was expressed in Sf9 insect cells, disrupted using nitrogen cavitation, and membranes harvested as described previously [45] . CD81 was expressed in Pichia pastoris yeast cells and membranes harvested as described previously [15] . For all proteins, membranes were resuspended in buffer 1 at 60 mg/ml (wet pellet weight) and stored in aliquots at −80 °C.…”

“…SMA can therefore extract membrane proteins directly from the membrane, to form a small soluble particle, with the protein retaining its lipid bilayer environment. SMA has been shown to work effectively for a wide range of different membrane proteins from many different expression systems [9] , [10] , [11] , [12] , [13] , [14] , [15] . SMALPs, once formed, are stable and excess free SMA can be removed.…”

Biological insights from SMA-extracted proteins

“…Thus, it seems more likely that lipid-interactions will be maintained when using SMA than conventional detergents. Several studies have reported analysis of the lipid content of purified protein SMALPs using techniques such as TLC [ 13 , 32 , 37 , 66 , 71 ], HPLC [ 54 ] and/or mass spectrometry [ 22 , 71 , 72 ]. Lipids co-purified from E.coli with SMA-extracted KcsA were found to be enriched in both cardiolipin (CL), and phosphatidylglycerol (PG) [ 13 ], both of which are negatively charged at physiological pH.…”

“…It is not yet clear whether SMA (or other polymers) has bias towards solubilising specific lipids. One study reported no differences in lipid content between SMALPs and the originating E. coli membrane [ 72 ], whereas subtle differences were observed between total membrane and SMA solubilised samples for Pichia pastoris expressing CD81 [ 22 ]. Lipidomic analysis of nanodiscs generated using a variety of polymers, including SMA and DIBMA, suggested that glycerolipids are strongly enriched and phospholipids de-enriched in polymer-solubilised samples compared with the originating E. coli and Jurkat membranes [ 75 ].…”

“…One of these is a sensitivity to pH below 7 or to divalent cations, which can be problematic for proteins which require more acidic conditions or divalent cation binding for function, such as ATP hydrolysis [18,19]. Another issue is encountered with free excess SMA which can interfere with effective binding to affinity resins or antibodies and other downstream procedures [15,[20][21][22]. As a result of these limitations, numerous polymer modifications are being investigated [23,24], including DIBMA (diisobutylene maleic acid), which has an aliphatic group in place of styrene, and has been shown to generate slightly larger discs, which are more tolerant to divalent cations [25,26].…”