Lactose permease (LacY) is the prototype of the major facilitator superfamily (MFS) of secondary transporters. Available structures of LacY reveal a state in which the substrate is exposed to the cytoplasm but is occluded from the periplasm. However, the alternating-access transport mechanism requires the existence of a periplasm-facing state. We recently showed that inverted-topology structural repeats provide the foundation for the mechanisms of two transporter families with folds distinct from the MFS. Here, we generated a structural model of LacY by swapping the conformations of inverted-topology repeats identified in its two domains. The model exhibits all required properties of an outward-facing conformation, i.e., closure of the binding site to the cytoplasm and exposure to the periplasm. Furthermore, the model agrees with double electron-electron resonance distance changes, accessibility to cysteine-modifying reagents, cysteine cross-linking data, and a recent structure of a distantly related transporter. Analysis of the intradomain differences between the two states suggests a role for conserved sequence motifs in occluding the central pathway through kinking of the pore-lining helices. In addition, predicted re-pairing of critical salt-bridging residues in the binding sites agrees remarkably well with previous proposals, allowing a description of the proton/sugar transport mechanism. More fundamentally, our model demonstrates that inverted-topology repeats provide the foundation for the alternating-access mechanisms of MFS transporters.
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