TRANSMEMBRANE HELIX TILTING AND LIGAND-INDUCED CONFORMATIONAL-CHANGESIN THE LACTOSE PERMEASE DETERMINED BY SITE-DIRECTED CHEMICAL CROSS-LINKING IN-SITU

The N-terminal six transmenbrane helices (N-6) and the C-terminal six transmembrane helices (C-6) of the lactose permease of Escherichia col i, each with a Cys residue, were co-expressed independently, and cross -linking was studied. Proximity of paired Cys residues in helices II ( position 49, 52, 53, 56, 57, 60, 63 or 67) and VII (position 227, 230, 231, 234, 238, 241, 242 or 245) or XI (position 350, 353, 354, 357, 3 61 or 364) was examined by using two homobifunctional thiol-specific c rosslinking agents of different lengths (6 or 10 Angstrom). The result s demonstrate that a Cys residue placed in the periplasmic half of hel ix II (position 49, 52, 53 or 57) crosslinks to Cys residues in the pe riplasmic half of helix VII (position 241, 242 or 245). In contrast, n o crosslinking is evident with paired-Cys residues in the cytoplasmic halves of helices LT (position 60, 63 or 67) and VII (position 227, 23 0, 231, 234 or 238). Remarkably, a Cys residue in the cytoplasmic half of helix II (position 60, 63 or 67) crosslinks with a Cys residue in the cytoplasmic half of helix XI (position 350, 353 or 354), while pai red-Cys residues at positions in the periplasmic halves of the two hel ices do not crosslink. Therefore, helix II is tilted in such a manner that the periplasmic end is close to helix VII, and the cytoplasmic en d is close to helix XI. Furthermore, ligand-binding alters the cross-l inking efficiency of paired-Cys residues in helices II and VII or XI, indicating that both interfaces are conformationally active. The resul ts are consistent with the conclusion that ligand-binding induces a sc issors-like movement of helices II and VII that increases interhelical distance by 3 to 4 Angstrom at the periplasmic ends and decreases the distance by 3 to 4 Angstrom at the approximate middle of the two tran smembrane helices. (C) 1998 Academic Press.