Investigating Protein-Lipid Interactions of MthK and Native Bacterial Membrane Lipids

Abstract

MthK is a Ca2+-activated bacterial potassium channel found in a wide range of prokaryotes. Previous electrophysiological and structural work focussed mainly on Ca2+-binding and the gating mechanism, while protein-lipid interactions of MthK are mostly unknown. Given that the eukaryotic homologue of MthK, the Big Potassium (BK) channel is known to be modulated by various lipids including Phosphatidylinositol-4,5-bisphosphate (PIP2), we investigated which lipids of bacterial membranes interact with MthK. The lipids used were 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 18:1 cardiolipin (CDL). We probed lipid binding through titration experiments using native mass spectrometry. These experiments showed a preferential binding of CDL over POPE and POPG to MthK. To assess specificity of these protein-lipid interactions coarse-grained molecular dynamics simulations of MthK in biomimetic membranes have been carried out. CDL binds in distinct binding pockets near the interface on the subunits while POPG shows unspecific binding to MthK. Further simulations are employed to probe the energetics of the binding events through Potential of Mean Force calculations and to comparing lipid interactions of MthK to lipid interactions in related channels including PIP2 binding to the BK channel.