Opening and closing of the periplasmic gate in lactose permease

X-ray crystal structures of lactose permease (LacY) reveal pseudosymmetrically 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 [rightward arrow] Cys/Asn-245 [rightward arrow] Cys, Thr-45 [rightward arrow] Cys/Asn-245 [rightward arrow] Cys, and Ile-32 [rightward arrow] Cys/Asn-245 [rightward arrow] 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 [almost equal to]15 Å 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 [almost equal to]15 Å in length. The results provide direct support for the argument that transport via LacY involves opening and closing of a large periplasmic cavity.