title: The alternating-access mechanism of MFS transporters arises from inverted-topology repeats.
abstract: 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.
authors.affiliation: Computational Structural Biology Group, Max Planck Institute of Biophysics, Max-von-Laue-Str. 3, 60438 Frankfurtam Main, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Radestock
authors.forename: Lucy R
authors.initials: LR
authors.lastname: Forrest
mesh_terms: D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005033:Ethylmaleimide / Q000737:chemistry; D008958:Models, Molecular; D009004:Monosaccharide Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012997:Solvents / Q000737:chemistry; D027981:Symporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; C088688:FucP protein, E coli; C050221:LacY protein, E coli; D009004:Monosaccharide Transport Proteins; D012997:Solvents; D027981:Symporters; D005033:Ethylmaleimide