Abstract
Membrane proteins play key roles in cellular functions, their activity mainly depending on their topological arrangement in membranes. Structural studies of membrane proteins have long adopted a protein-centric view regarding the determinants of membrane protein topology and function. Several studies have shown that the orientation of transmembrane domains of polytopic membrane proteins with respect to the plane of the lipid bilayer can be largely determined by membrane lipid composition. However, the mechanism by which membrane proteins exhibit structural and functional duality in the same membrane or different membranes is still unknown. Here we show that lipid-dependent structural and functional assessment of a membrane protein can be conducted in detergent micelles, opening the possibility for the determination of lipid-dependent high-resolution crystal structures. We found that the lactose permease purified from Escherichia coli cells exhibiting varied phospholipid compositions exhibits the same topology and similar function as in its membrane of origin. Furthermore, we found several conditions, including protein mutations and micelle lipid composition, that lead to increased protein stability, correlating with a higher yield of two-dimensional crystal formation. Altogether, our results demonstrate how the membrane lipid environment influences membrane protein topology and arrangement, both in native membranes and in mixed detergent micelles.
Highlights
Membrane proteins represent about 40% of the proteome and are essential components of many indispensable biological processes
LacY from Escherichia coli is a paradigm for secondary transporters throughout nature and has been heavily studied for many years[44]
The inverted topology of LacY observed in cells and proteoliposomes lacking the major membrane phospholipid PE (Fig. 1A) exhibits a similar yet different architecture compared to the wild type (WT) LacY and should be considered as a conformationally and topologically different yet related protein, posing significant new challenges for crystallization
Summary
Membrane proteins represent about 40% of the proteome and are essential components of many indispensable biological processes. Using several secondary transporters found in Escherichia coli including lactose permease (LacY) as model membrane proteins, we challenged this long-standing protein-centric dogma by demonstrating that the topological organization of a membrane protein, once established, is not static but is dynamic in response to changes in the lipid environment both in vivo and in vitro[27,28,29,30,31,32,33]. Membrane proteins can undergo TMD flipping after initial assembly in response to changes in the lipid environment in vivo[29,34,35,36,37] and in vitro[38,39,40,41] These two topological conformers are fully interconvertible post-assembly by the addition or deletion of PE. Membrane protein topological organization is not static but highly dynamic
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