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The lactose permease (LacY) of Escherichia coli is the prototype of the major facilitator superfamily, one of the largest families of membrane transport proteins. Structurally, two pseudo-symmetrical six-helix bundles surround a large internal aqueous cavity. Single binding sites for galactoside and H + are positioned at the approximate center of LacY halfway through the membrane at the apex of the internal cavity. These features enable LacY to function by an alternating-access mechanism that can catalyze galactoside/H + symport in either direction across the cytoplasmic membrane. The H + -binding site is fully protonated under physiological conditions, and subsequent sugar binding causes transition of the ternary complex to an occluded intermediate that can open to either side of the membrane. We review the structural and functional evidence that has provided new insight into the mechanism by which LacY achieves active transport against a concentration gradient. 

The lactose permease ofEscherichia coli(LacY) utilizes an alternating access symport mechanism with multiple conformational intermediates, but only inward (cytoplasmic)- or outward (periplasmic)-open structures have been characterized by X-ray crystallography. It is demonstrated here with sugar-binding studies that cross-linking paired-Cys replacements across the closed cytoplasmic cavity stabilize an occluded conformer with an inaccessible sugar-binding site. In addition, a nanobody (Nb) that stabilizes a periplasmic-open conformer with an easily accessible sugar-binding site in WT LacY fails to cause the cytoplasmic cross-linked mutants to become accessible to galactoside, showing that the periplasmic cavity is closed. These results are consistent with tight association of the periplasmic ends in two pairs of helices containing clusters of small residues in the packing interface between N- and C-terminal six-helix bundles of the symporter. However, after reduction of the disulfide bond, the Nb markedly increases the rate of galactoside binding, indicating unrestricted access to the Nb epitope and the galactoside-binding site from the periplasm. The findings indicate that the cross-linked cytoplasmic double-Cys mutants resemble an occluded apo-intermediate in the transport cycle. 

Lactose permease is a paradigm for the major facilitator superfamily, the largest family of ion-coupled membrane transport proteins known at present. LacY carries out the coupled stoichiometric symport of a galactoside with an H⁺, using the free energy released from downhill translocation of H⁺to drive accumulation of galactosides against a concentration gradient. In neutrophilicEscherichia coli, internal pH is kept at ∼7.6 over the physiological range, but the apparent pK (pK^app) for galactoside binding is 10.5. Surface-enhanced infrared absorption spectroscopy (SEIRAS) demonstrates that the high pK_ais due to Glu325 (helix X), which must be protonated for LacY to bind galactoside effectively. Deprotonation is also obligatory for turnover, however. Here, we utilize SEIRAS to study the effect of mutating residues in the immediate vicinity of Glu325 on its pK_a. The results are consistent with the idea that Arg302 (helix IX) is important for deprotonation of Glu325. 

The lactose permease of Escherichia coli (LacY), a dynamic polytopic membrane transport protein, catalyzes galactoside/H + symport and operates by an alternating access mechanism that exhibits multiple conformations, the distribution of which is altered by sugar-binding. Camelid nanobodies were made against a double-mutant Gly46 → Trp/Gly262 → Trp (LacY WW ) that produces an outward-open conformation, as opposed to the cytoplasmic open-state crystal structure of WT LacY. Nanobody 9047 (Nb9047) stabilizes WT LacY in a periplasmic-open conformation. Here, we describe the X-ray crystal structure of a complex between LacY WW , the high-affinity substrate analog 4-nitrophenyl-α- d -galactoside (NPG), and Nb9047 at 3-Å resolution. The present crystal structure demonstrates that Nb9047 binds to the periplasmic face of LacY, primarily to the C-terminal six-helical bundle, while a flexible loop of the Nb forms a bridge between the N- and C-terminal halves of LacY across the periplasmic vestibule. The bound Nb partially covers the vestibule, yet does not affect the on-rates or off-rates for the substrate binding to LacY WW , which implicates dynamic flexibility of the Nb–LacY WW complex. Nb9047-binding neither changes the overall structure of LacY WW with bound NPG, nor the positions of side chains comprising the galactoside-binding site. The current NPG-bound structure exhibits a more occluded periplasmic vestibule than seen in a previous structure of a (different Nb) apo-LacY WW /Nb9039 complex that we argue is caused by sugar-binding, with major differences located at the periplasmic ends of transmembrane helices in the N-terminal half of LacY.