Membrane Remodeling by GltPh in the Inward- and Outward-Facing Conformations Explains Lack of Protomer Cooperativity

Abstract

Membrane-embedded proteins can induce the remodeling of the adjacent lipid bilayers by promoting curvature, altering the membrane thickness and/or exposing hydrophobic groups. When these proteins undergo a function-related conformational transition, the energy cost associated with these membrane perturbations adds up to the free energy of each of the protein states and can therefore modulate their functional mechanisms. Here, we study the case of the Na+-coupled aspartate transporter from Pyrococcus horikoshii (GltPh). GltPh has been crystallized as a trimer both in an inward- and outward-facing conformations. In this outward-to-inward conformational exchange, the so-called transport domain moves ∼20 A across the membrane, relative to the seemingly rigid trimerization/scaffold domains, so as to expose the substrate and Na+-binding sites to either the cytoplasm or the extracellular space. Using large-scale coarse-grained and all-atom molecular dynamics (MD) simulations, we characterized the membrane deformation induced by GltPh trimers in all possible permutations of inward-facing and outward-facing protomer states. The simulations show that when a protomer is in the outward-facing state, the surrounding membrane is largely unperturbed. However, in the inward-facing state, the transport domain induces a strong deformation on the lipid bilayer, bending its average plane by ∼10 A along the direction perpendicular to the membrane plane. This perturbation extends radially for ∼50 A, but remarkably, it is largely localized around each of the transport domains in the vicinity of the protein, i.e. the membrane shape is restored near the protomer-protomer interfaces. The specific protein-lipid contacts that explain this local deformation are identified from all-atom MD simulations. In summary, MD simulations demonstrate that the lipid membrane favors the outward-facing state of GltPh over the inward-facing state, owing to the long-range curvature deformation induced by the latter. However, these simulations also show that this large deformation does not imply a membrane-mediated protomer cross-talk, explaining the mystifying absence of measurable cooperativity among protomers in this trimeric transporter.