(493b) Regulation of Multispanning Membrane Protein Topology Via Kinetic Annealing of the Stop-Translation Ensemble

The topology of a multispanning membrane protein is typically expected to be established during cotranslational membrane integration such that the orientation of the N-terminal transmembrane domain dictates the overall topology of the protein with respect to the cell membrane. However, this mechanism is inconsistent with the behavior of dual-topology proteins for which the overall topology of the protein can be biased via mutation of charged residues on the soluble loops.  In particular, EmrE is a dual-topology protein for which the inclusion of positive loop charges, even close to the C-teminus, leads to dramatic shifts in its overall topology. In this work, we use a recently developed coarse-grained simulation model of Sec-facilitated membrane integration to investigate this behavior in EmrE and its mutants. Excellent agreement is obtained between the membrane protein topologies predicted from the simulations and those observed experimentally, suggesting that the simulations capture the underlying molecular processes that govern topological shifts. We find that, in contrast to some single-topology multispanning proteins, a large fraction of the transmembrane domains of EmrE and its mutants are misintegrated at the end of ribosomal translation. These misintegrated configurations subsequently anneal to fully integrated multispanning configurations via the flipping of soluble loops across the membrane. The energetic barriers associated with flipping the soluble loops enforce kinetic pathways that dictate the final topology of the fully integrated protein. This proposed mechanism, in which kinetic annealing of the stop-translation ensemble dictates the topology of multispanning proteins, is consistent with a range of experimentally observed features of the topogenesis of EmrE and its mutants; furthermore, the mechanism offers new experimentally-testable predictions regarding the effect of translocon mutations on topogenesis. This work provides insight into the topogenesis of dual-topology proteins, with additional relevance to the topogenesis and folding of broader classes of multispanning membrane proteins.