RESUMO
Over two-thirds of integral membrane proteins of known structure assemble into oligomers. Yet, the forces that drive the association of these proteins remain to be delineated, as the lipid bilayer is a solvent environment that is both structurally and chemically complex. In this study, we reveal how the lipid solvent defines the dimerization equilibrium of the CLC-ec1 Cl-/H+ antiporter. Integrating experimental and computational approaches, we show that monomers associate to avoid a thinned-membrane defect formed by hydrophobic mismatch at their exposed dimerization interfaces. In this defect, lipids are strongly tilted and less densely packed than in the bulk, with a larger degree of entanglement between opposing leaflets and greater water penetration into the bilayer interior. Dimerization restores the membrane to a near-native state and therefore, appears to be driven by the larger free-energy cost of lipid solvation of the dissociated protomers. Supporting this theory, we demonstrate that addition of short-chain lipids strongly shifts the dimerization equilibrium toward the monomeric state, and show that the cause of this effect is that these lipids preferentially solvate the defect. Importantly, we show that this shift requires only minimal quantities of short-chain lipids, with no measurable impact on either the macroscopic physical state of the membrane or the protein's biological function. Based on these observations, we posit that free-energy differentials for local lipid solvation define membrane-protein association equilibria. With this, we argue that preferential lipid solvation is a plausible cellular mechanism for lipid regulation of oligomerization processes, as it can occur at low concentrations and does not require global changes in membrane properties.
A cell's outer membrane is made of molecules called lipids, which band together to form a flexible thin film, just two molecules thick. This membrane is dotted with proteins that transport materials in to and out of cells. Most of these membrane proteins join with other proteins to form structures known as oligomers. Except, how membrane-bound proteins assemble into oligomers the physical forces driving these molecules to take shape remains unclear. This is partly because the structural, physical and chemical properties of fat-like lipid membranes are radically different to the cell's watery interior. Consequently, the conditions under which membrane oligomers form are distinct from those surrounding proteins inside cells. Membrane proteins are also more difficult to study and characterize than water-soluble proteins inside the cell, and yet many therapeutic drugs such as antibiotics specifically target membrane proteins. Overall, our understanding of how the unique properties of lipid membranes affect the formation of protein structures embedded within, is lacking and warrants further investigation. Now, Chadda, Bernhardt et al. focused on one membrane protein, known as CLC, which tends to exist in pairs or dimers. To understand why these proteins form dimers (a process called dimerization) Chadda, Bernhardt et al. first used computer simulations, and then validated the findings in experimental tests. These complementary approaches demonstrated that the main reason CLC proteins 'dimerize' lies in their interaction with the lipid membrane, and not the attraction of one protein to its partner. When CLC proteins are on their own, they deform the surrounding membrane and create structural defects that put the membrane under strain. But when two CLC proteins join as a dimer, this membrane strain disappears making dimerization the more stable and energetically favorable option. Chadda, Bernhardt et al. also showed that with the addition of a few certain lipids, specifically smaller lipids, cell membranes become more tolerant of protein-induced structural changes. This might explain how cells could use various lipids to fine-tune the activity of membrane proteins by controlling how oligomers form. However, the theory needs to be examined further. Altogether, this work has provided fundamental insights into the physical forces shaping membrane-bound proteins, relevant to researchers studying cell biology and pharmacology alike.
