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 --> 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 approximately 15 A 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 approximately 15 A in length. The results provide direct support for the argument that transport via LacY involves opening and closing of a large periplasmic cavity.
Zhou
Yonggang
Y
Departments of Physiology and Microbiology, Immunology, and Molecular Genetics, Molecular Biology Institute, University of California, Los Angeles, CA 90095-1662, USA.
Guan
Lan
L
Freites
J Alfredo
JA
Kaback
H Ronald
HR
D001692:Biological Transport / Q000187:drug effects; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000494:pharmacology; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology*; D007785:Lactose / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D008968:Molecular Conformation; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D019897:Periplasm / Q000187:drug effects / Q000378:metabolism*
D003432:Cross-Linking Reagents; D026901:Membrane Transport Proteins; D050505:Mutant Proteins; C021499:lactose permease; D007785:Lactose