A Mass Spectrometric Platform to Reveal Lipid Mediated Oligomerisation of Membrane Proteins in Atomistic Detail

Abstract

The structural and functional effects resulting from the interactions between membrane proteins (MP) and their surrounding lipids is a subject of constant debate. A major facet of these discussions is the role of lipids in regulating the oligomeric states of MP assemblies. To date, no multimeric MP structures exist in the PDB with lipids at the protein-protein interface. This highlights the limitation of crystallography in determining the identity of lipid molecules, owing to their conformational flexibility and high mobility. Exploiting the ability of native-mass spectrometry (nMS) to simultaneously observe the binding of lipids to MP and their impact on oligomeric states we have developed for the first time a model for lipid induced oligomerisation in MP in atomistic detail and set up a general platform to study this phenomenon in other MPs. An extensive bioinformatics analysis of all MPs led to a set of model systems with weak interfaces, potentially suggesting lipid mediation. nMS analysis of these MPs revealed a defined mass addition to the oligomer. Through an integrative lipid-analysis, using a novel high-energy platform that allows MS/MS analysis of MPs, we identified bound phospholipids and cardiolipins. Analysis of delipidated proteins, as well as mutants that lack dimeric interactions and the probable lipid binding sites, proposed through MD simulation, coupled with integrated lipid analysis and live cell imaging, revealed an atomistic model of how lipids can act as plug to bridge weekly bound oligomers. The approach, relying on the unique ability of MS to determine the oligomeric states of MPs, with precise identification of bound endogenous lipids, provides a platform that will further the understanding of the process of lipid-induced oligomerisation in MPs.