Sugar recognition by the lactose permease of Escherichia coli.
Biochemical, luminescence and mass spectroscopy approaches indicate that Trp-151 (helix V) plays an important role in hydrophobic stacking with the galactopyranosyl ring of substrate and that Glu-269 (helix VIII) is essential for substrate affinity and specificity. The x-ray structure of the lactose permease (LacY) with bound substrate is consistent with these conclusions and suggests that a possible H-bond between Glu-269 and Trp-151 may play a critical role in the architecture of the binding site. We have now probed this relationship by exploiting the intrinsic luminescence of a single Trp-151 LacY with various replacements for Glu-269. Mutations at position 269 dramatically alter the environment of Trp-151 in a manner that correlates with binding affinity of LacY substrates. Furthermore, chemical modification of Trp-151 with N-bromosuccinimide indicates that Glu-269 forms an H-bond with the indole N. It is concluded that 1) an H-bond between the indole N and Glu-269 optimizes the formation of the substrate binding site in the inward facing conformation of LacY, and 2) the disposition of the residues implicated in sugar binding in different conformers suggests that sugar binding by LacY involves induced fit.
Vázquez-Ibar
José Luis
JL
Department of Physiology and Microbiology, Molecular Biology Institute, Howard Hughes Medical Institute, Los Angeles, CA 90095-1662, USA.
Guan
Lan
L
Weinglass
Adam B
AB
Verner
Gill
G
Gordillo
Ruth
R
Kaback
H Ronald
HR
D001665:Binding Sites; D001692:Biological Transport; D001974:Bromosuccinimide / Q000494:pharmacology; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D018698:Glutamic Acid / Q000737:chemistry; D006860:Hydrogen Bonding; D007700:Kinetics; D013058:Mass Spectrometry; D026901:Membrane Transport Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D008954:Models, Biological; D008958:Models, Molecular; D009154:Mutation; D010100:Oxygen / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013379:Substrate Specificity; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry
D026901:Membrane Transport Proteins; D018698:Glutamic Acid; D014364:Tryptophan; C021499:lactose permease; D001974:Bromosuccinimide; D010100:Oxygen