Lipid modulation of membrane protein conformational equilibria does not require lipid immobilization in long-lasting protein-lipid complexes

Lipid regulation of membrane proteins is widely rationalized as a process whereby lipid molecules become immobilized in a complex with the protein, supposedly due to strong, specific interactions. While this conventional paradigm might apply to some regulatory processes, we posit that it is not a universal mechanism. As a counterexample, we examine the mechanism by which lipids modulate the dimerization reaction of the bacterial CL−/H+ antiporter CLC-ec1. Using molecular simulations, we have shown that monomeric CLC-ec1 induces a pronounced defect in the morphology of the surrounding membrane, including thinning of the hydrophobic core due to drastic lipid tilting and entanglement of the two bilayer leaflets. Upon dimerization, this interface becomes buried, and the morphological defect in the membrane is completely eliminated. Using advanced simulation methods, we have shown that the free-energy gain resulting from the elimination of this defect accounts for much of the free-energy of CLC-ec1 association measured experimentally. These experiments, based on single-molecule microscopy, also demonstrate that introduction of short-chain lipids drastically destabilize dimerization, but the concentration dependence of this inhibitory effect is incoherent with a conventional competitive ligand-binding process. To examine the microscopic mechanism underlying these intriguing observations, we calculate a continuous 40-microsecond molecular dynamics trajectory of all-atom monomeric CLC-ec1 embedded in a 70:30 mixture of POPC and DLPC lipids. This simulation shows that DLPC molecules become enriched within the thinned-membrane defect, alleviating its energetic cost and hence inhibits dimerization. However, we find that this enrichment does not stem from the immobilization of these lipids in a long-lasting protein-lipid complex, but from subtle yet statistically significant differences in the residence times and exchange frequency of PO and DL lipids within the first solvation shells.