Regulation of Multispanning Membrane Protein Topology Via the Post-Translational Flipping of Charged Protein Elements

Multispanning membrane proteins – i.e., proteins with multiple transmembrane domains that thread back-and-forth across the cell membrane - are essential for cellular functions that include signal transduction, material transport, and energy conversion. Performing these functions requires the proteins to integrate within the membrane in the correct topology, or the overall orientation of the protein relative to the membrane. Conventionally, it is believed that a single membrane protein topology is determined during translation by the orientation of the first transmembrane segment that integrates into the membrane. Challenging this view, the multispanning, dual-topology protein EmrE obtains two possible topologies with equal likelihood. Moreover, the topology of EmrE is highly sensitive to single charge mutations, including C-terminal mutations, indicating that topology is adjustable well after the onset of translation. Understanding the origin of this topological plasticity could yield new insight into membrane protein structure and function, as well as providing guidelines for engineering proteins for specific topological orientations.

In this work, we use coarse-grained simulations to investigate the integration of the multispanning membrane protein EmrE on realistic biological timescales. We employ a simulation model that enables access to a timescale of minutes while retaining sufficient chemical accuracy to capture the forces that drive membrane integration. We find that EmrE establishes its topology post-translationally by the stochastic “flipping” of charged protein elements across the membrane over long time scales. This behavior is surprising because the transport of charged moieties across the membrane is thought to occur on timescales too long to be biologically relevant. We further use atomistic molecular dynamics simulations to demonstrate that such timescales can be dramatically reduced by cooperative interactions between amino-acid side chains that minimize the free energy barrier for flipping. Together, these results suggest a new mechanism for multispanning membrane protein topogenesis and indicate that post-translational topological rearrangements may play an important role in determining membrane protein structure and function.