Mechanistic and thermodynamic characterization of dynamic topology in an unassembled transmembrane protein

It is conventionally believed that multispanning membrane protein topology (the orientation of the protein relative to the membrane) is established during protein synthesis through the process of cotranslational membrane integration. However, this mechanism is inconsistent with the behavior of EmrE, a dual-topology protein for which the N-terminal transmembrane helix flips in and out of the membrane on physiologically relevant timescales. Experiments have shown that flipping occurs after protein synthesis when EmrE is an unassembled monomer and is arrested upon dimerization, although the molecular mechanisms underlying these observations remain unknown. In this work, we use atomistic molecular dynamics simulations and enhanced sampling to investigate the thermodynamics of this flipping process. The simulations reveal a mechanism for enhancing the flipping of the N-terminal transmembrane helix of EmrE, in which a charged, membrane-exposed glutamate residue (E14) at the center of the helix lowers energetic barriers to flipping by decreasing flipping-induced perturbations to lipid bilayer structure. Analysis of interhelical hydrogen bonding patterns further shows that interactions between EmrE monomers upon dimerization leads to the stabilization of the structure and topology of the EmrE dimer to inhibit flipping. The proposed mechanism highlights the critical role of E14 in regulating the topological stability of EmrE. Together, our results reveal new molecular-scale insight into processes by which specific sequence features (i.e., membrane-exposed charged residues) can promote post-translational changes to membrane protein topology.