Helix proximity and ligand-induced conformational changes in the lactose permease of Escherichia coli determined by site-directed chemical crosslinking.
N and C-terminal halves of lactose permease, each with a single-Cys residue, were co-expressed, and crosslinking was studied. Iodine or N,N'-o-phenylenedimaleimide (o-PDM; rigid 6 A), crosslinks Asn245 Cys (helix VII) and Ile52 --> Cys or Ser53 --> Cys (helix II). N,N'-p-phenylenedimaleimide (p-PDM; rigid 10 A) crosslinks the 245/53 Cys pair weakly, but does not crosslink 245/52, and 1,6-bis-maleimidohexane (BMH; flexible 16 A) crosslinks both pairs less effectively than o-PDM. Thus, 245 is almost equidistant from 52 and 53 by up to about 6 A. BMH or p-PDM crosslinks Gln242 --> Cys and Ser53 --> Cys, but o-PDM is ineffective, indicating that distance varies by up to 10 A. Ligand binding increases crosslinking of 245/53 with p-PDM or BMH, has little effect with o-PDM and decreases iodine crosslinking. Similar effects are observed with 245/52. Ligand increases 242/53 crosslinking with p-PDM or BMH, but no crosslinking is observed with o-PDM. Therefore, ligand induces a translational or scissors-like displacement of the helices by 3-4 A. Crosslinking 245/53 inhibits transport indicating that conformational flexibility is important for function.
Wu
J
J
Department of Physiology, University of California, Los Angeles 90095-1662, USA.
Kaback
H R
HR
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001692:Biological Transport; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D003545:Cysteine / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology* / Q000276:immunology; D029968:Escherichia coli Proteins; D008024:Ligands; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation / Q000187:drug effects*; D017433:Protein Structure, Secondary / Q000187:drug effects; D027981:Symporters
D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D008024:Ligands; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; C021499:lactose permease; D003545:Cysteine