Membrane transporter dimerization driven by differential lipid solvation energetics of dissociated and associated states

Abstract

Over two-thirds of membrane proteins of known structure assemble into oligomers. Yet, the forces that drive the association of these proteins in the membrane 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 an energetic penalty for solvating a thinned-membrane defect caused by their exposed dimerization interfaces. Supporting this theory, we demonstrate that this penalty is drastically reduced with minimal amounts of short-chain lipids, which stabilize the monomeric state by preferentially solvating the defect rather than altering the physical state of the membrane. We thus posit that the energy differentials for local lipid-solvation define membrane-protein association equilibria, and describe a molecular-level physical mechanism for lipid regulation of such processes in biological conditions.

Dimerization mechanism of an inverted-topology ion channel in membranes

“…While Na + binding is required for activation, single-molecule photobleaching analysis reveals that the protein dimerizes with strong affinity, ΔG° = -10.3 ± 0.4 kcal/mole, 1 subunit/lipid standard state, in the absence of Na + ; Na + binding to the dimer state is only weakly stabilizing. Coarse-grained molecular dynamics simulations of the Fluc monomer in membranes, shows that the lipid bilayer forms a thinned and curved defect around the dimerization interface, similar to what we observed previously in our studies of CLC (9). Finally, simulations of freely diffusing monomers in the membrane, designed to preclude direct protein-protein contacts, nevertheless show that Fluc selectively forms stable dimers in the native orientation, which bury the membrane defect.…”

“…To investigate this mechanism and the nature of this driving force, we first carried out coarse-grained molecular dynamics (CGMD) simulations of monomeric and dimeric Fluc in 2:1 POPE/POPG membranes. The calculated trajectories are 50-µs long, which allows for complete mixing of the two lipid species as well as multiple turnovers of all lipid molecules in the protein solvation shells in exchange for other lipids in the bulk (9). Inspection of the membrane morphology in these trajectories, through 2D maps of the hydrophobic thickness, shows the monomer induces a thinned-membrane defect at the exposed dimerization interface, which disappears upon dimerization (Fig.…”

“…Aside from model systems, the only integral membrane transport protein whose assembly thermodynamics has been characterized is the CLC-ec1 Cl -/H + antiporter, a homologue to voltage-gated chloride ion channels in humans. This transporter has been shown to exist in a reversible homo-dimerization equilibrium in membranes (8), largely controlled by differentials in the lipid solvation energetics of the monomeric and dimeric states (9). Reversible oligomerization of membrane proteins occurs also beyond the channel family; a remarkable example is the bacterial flagellar motor, whose membrane-embedded subunits have been shown to be in continuous exchange, even when the motor is active (10).…”

Lipid Bilayer Induces Contraction of the Denatured State Ensemble of a Helical-Bundle Membrane Protein

“…The copyright holder for this preprint (which this version posted May 17, 2021. ; https://doi.org/10.1101/2021.05.17.444377 doi: bioRxiv preprint Lipophobic effect depends on the context of native structure and lipid environment. While we propose that the lipophobic effect generally induces protein compaction in a lipid bilayer, recent studies indicate that this solvent-dependent effect occurs through multiple mechanisms that can be modulated by the native structure and lipid environment: Mode 1) Lipid deformation at the protein surface (71): For example, in the dynamic monomerdimer equilibrium of the CLC-ec1 Cl -/H + antiporter, a thicker lipid bilayer induces a hydrophobic mismatch between the dimer interface on each monomer and the bilayer, resulting in local thinning of the bilayer by deformation of the lipid molecules at the interface (72). This local thinning is pronounced in the monomers and shifts the equilibrium towards dimers, which can be overcome by the incorporation of lipids with shorter acyl chains that preferentially solvate the interface (72), Mode 2) Exposure of the polar residues in the bilayer: The isolated N-and C-domains in each CLC-ec1 monomer associate with more water molecules than the folded monomer at the domain interface due to the exposure of the polar residues in the bilayer core (73) We find that the DSE of GlpG expands more in E. coli liposomes than in bicelles despite a similar quasi-2D physical constraint.…”

“…Lipid deformation at the protein surface (71): For example, in the dynamic monomerdimer equilibrium of the CLC-ec1 Cl -/H + antiporter, a thicker lipid bilayer induces a hydrophobic mismatch between the dimer interface on each monomer and the bilayer, resulting in local thinning of the bilayer by deformation of the lipid molecules at the interface(72). This local thinning is pronounced in the monomers and shifts the equilibrium towards dimers, which can be overcome by the incorporation of lipids with shorter acyl chains that preferentially solvate the interface (72), Exposure of the polar residues in the bilayer: The isolated N-and C-domains in each CLC-ec1 monomer associate with more water molecules than the folded monomer at the domain interface due to the exposure of the polar residues in the bilayer core(73).The energetic penalty caused by the hydration of the membrane-buried domain interface can drive the folding of each monomer (73), Mode 3) Chemical and physical properties of the bulk lipid bilayer: TM helix-helix interactions can be modulated by lipid composition with negligible changes in the bilayer thickness for various types of single membrane-spanning helices, including the TM domain of glycophorin A (74, 75), a proton channel M2 (76), a transcriptional regulator Mga2 (77) and an endoplasmic reticulum stress sensor Ire1a (78).…”