(587e) Mechanistic and Thermodynamic Characterization of Dynamic Membrane Topology in an Unassembled Membrane Protein

Bacterial transmembrane proteins of the small multidrug resistance (SMR) family have been extensively studied for their role in expelling drug-like molecules. EmrE, a homodimeric SMR transporter, has four transmembrane helices (TMHs) in each monomer. EmrE is also unusual in that it is a dual topology protein: its two monomers insert with opposite orientations in the membrane despite having the same sequence. Moreover, the N-terminal transmembrane helix of EmrE has been experimentally shown to flip in and out of the membrane on physiologically relevant timescales after co-translational membrane insertion as an unassembled monomer, which may be necessary for the dual-topology behavior of EmrE. This dynamic topology of EmrE monomers is intriguing since flipping hydrophilic residues across the hydrophobic core of the membrane requires high free energy cost, raising questions about the underlying molecular mechanisms by which the N-terminal helix flips.

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 helix of EmrE in which a charged residue (GLU14) at the center of the helix lowers energetic barriers to flipping by decreasing perturbations to lipid bilayer structure. Analysis of interhelical hydrogen bonding patterns shows that dimerization leads to the stabilization of the structure and topology of the EmrE dimer to inhibit further flipping. The proposed mechanism highlights the critical role of GLU14 in regulating the topological stability of EmrE, whose protonation state determines perturbations of membrane structure and interhelical H-bond patterns. Together, our results reveal new molecular-scale insight into processes by which specific sequence features (i.e., interhelical charged residues) can promote post-translational changes to membrane protein topology, which both have relevance to understanding the topology of EmrE and engineering similar flipping behavior into other proteins.