Structural characterisation of human tetraspanins and their interaction with cholesterol and gangliosides

Abstract

Tetraspanins are a family of membrane proteins that play a role in various functions, such as cell migration, signal transduction and intracellular trafficking. They are organisers within the membrane, forming tetraspanin-enriched microdomains (TEMs) comprised of tetraspanins, partner proteins, cholesterol and gangliosides. Knowledge of the precise interactions between these molecules is limited. Understanding more about these interactions would add to the body of knowledge about how TEMs are formed. With this knowledge these interactions could be disrupted to prevent negative biological events associated with TEMs, such as microbial infections. To study these interactions CD81, which interacts with hepatitis C virus, was used as a model tetraspanin. The purification of CD81 expressed in Pichia pastoris in styrene-maleic acid lipid particles (SMALPs) was optimised by including imidazole in the binding buffer and increasing the concentration of imidazole in the purification wash steps. CD81 mutants were created to study cholesterol-dependent conformational change using electron paramagnetic resonance. Protein-ligand docking was used to investigate the interaction between the two tetraspanins, CD81 and CD82, and the sugar residues in gangliosides. Asp122 in CD81 and Asp37 and Ser135 in CD82 were identified as interaction sites. The basic structure of tetraspanins appears to be ubiquitous but a variable region in the large extracellular loop (LEL) requires further research because this is a crucial area for tetraspanin interactions. LELs of some human tetraspanins were modelled and their disulfide bond arrangement was analysed, finding a different arrangement in the subset of tetraspanins referred to as TspanC6-CxCs. Sequence and structural alignment of human tetraspanins highlighted conserved residues within different structural regions that were used as anchor residues to build a universal amino acid numbering system.