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

Hoor Ayub,Michelle Clare,Ivana Milic,Nikola P Chmel,Heike Böning,Andrew Devitt,Thomas Krey,Roslyn M Bill,Alice J Rothnie

doi:10.1016/j.bbamem.2020.183419

Hoor Ayub, Michelle Clare + Show 7 more

Open Access

https://doi.org/10.1016/j.bbamem.2020.183419

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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.

Highlights

CD81 is a member of the tetraspanin family of membrane proteins that have broad biological and medical importance
Having established that styrene-maleic anhydride co-polymers (SMA) polymers were capable of effectively extracting CD81 from P. pastoris membranes, the step was to optimise the conditions for this extraction
As no difference was observed in the abilities of SMA 2000 and SZ25010 to extract CD81 (Figure 1), and previous reports with other proteins suggested SZ25010 gives lower stability and yield in downstream purification, it was decided to proceed solely with SMA 2000, which had previously been shown to be the best polymer to date [36]

Summary

Introduction

CD81 is a member of the tetraspanin family of membrane proteins that have broad biological and medical importance. Tetraspanins engage in a wide range of incompletelyunderstood molecular interactions, exerting biological functions in cell-cell adhesion, cell proliferation, the immune system and during fertilization. They coordinate the trafficking of molecules into tetraspanin-enriched membrane microdomains thereby affecting cell signaling, morphology, motility and fusion [1]. There are 34 tetraspanin family members in mammals, of which 33 have been identified in humans [1]. Tetraspanin proteins have four transmembrane domains (TM1-4), intracellular N- and C-termini and two extracellular domains, one small (known as EC1 or SEL) and one large (typically 100 residues; known as EC2 or LEL). The 6 Å cryo-electron microscopy structure of a uroplakin tetraspanin revealed a rod-shaped structural morphology consisting of four TM helical bundles bound to a single TM helix partner [6]

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