Opening and closing of the periplasmic gate in lactose permease

X-ray crystal structures of lactose permease (LacY) reveal pseudo-symmetrically arranged N- and C-terminal six-transmembrane helix bundles surrounding a deep internal cavity open on the cytoplasmic side and completely closed on the periplasmic. side. The residues essential for sugar recognition and H+ translocation are located at the apex of the cavity and are inaccessible from the outside. On the periplasmic side, helices I/II and VII from the N- and C- six helix bundles, respectively, participate in sealing the cavity from the outside. Three paired double-Cys mutants-Ile-40 -> Cys/Asn-245 -> Cys, Thr-45 -> Cys/Asn-245 -> Cys, and Ile-32 -> Cys/Asn-245 -> Cys-located in the interface between helices I/II and VII on the periplasmic side of LacY were constructed. After cross-linking with homobifunctional reagents less than approximate to 15 angstrom in length, all three mutants lose the ability to catalyze lactose transport. Strikingly, however, full or partial activity is observed when cross-linking is mediated by flexible reagents greater than approximate to 15 angstrom in length. The results provide direct support for the argument that transport via LacY involves opening and closing of a large periplasmic cavity.