Helix proximity and ligand-induced conformational changes in the lactose permease of Escherichia coli determined by site-directed chemical crosslinking

• Published in: Journal of molecular biology Vol. 270; no. 2; pp. 285 - 293
• Main Authors: Wu, Jianhua, Kaback, H.Ronald
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 11.07.1997
• Subjects: Amino Acid Sequence; Antibodies, Monoclonal - pharmacology; Biological Transport; conformational change; Cross-Linking Reagents - pharmacology; crosslinking; Cysteine - chemistry; Escherichia coli - enzymology; Escherichia coli - immunology; Escherichia coli Proteins; helix packing; Ligands; Membrane Transport Proteins - chemistry; Membrane Transport Proteins - immunology; Membrane Transport Proteins - metabolism; Monosaccharide Transport Proteins; Mutagenesis, Site-Directed; Protein Conformation - drug effects; Protein Structure, Secondary - drug effects; split permease; Symporters; transport; p-PDM; TDG; HRP; o-PDM; transport; conformational change; BMH; crosslinking; helix packing; split permease; mAb
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1006/jmbi.1997.1099

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 Å), crosslinks Asn245→Cys (helix VII) and Ile52→Cys or Ser53→Cys (helix II). N, N′- p-phenylenedimaleimide ( p-PDM; rigid 10 Å) crosslinks the 245/53 Cys pair weakly, but does not crosslink 245/52, and 1,6- bis-maleimidohexane (BMH; flexible 16 Å) crosslinks both pairs less effectively than o-PDM. Thus, 245 is almost equidistant from 52 and 53 by up to about 6 Å. BMH or p-PDM crosslinks Gln242→Cys and Ser53→Cys, but o-PDM is ineffective, indicating that distance varies by up to 10 Å. 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 Å. Crosslinking 245/53 inhibits transport indicating that conformational flexibility is important for function.