期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 115, 期 16, 页码 4146-4151出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1800706115
关键词
membrane transport proteins; lactose permease; fluorescence; nanobodies; stopped; flow
资金
- NIH [GM120043]
- National Science Foundation Eager Grant [MCB1747705]
- Canadian Glycomics Network of Centres of Excellence
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [R01GM120043] Funding Source: NIH RePORTER
Binding kinetics of alpha-galactopyranoside homologs with fluorescent aglycones of different sizes and shapes were determined with the lactose permease (LacY) of Escherichia coli by FRET from Trp151 in the binding site of LacY to the fluorophores. Fast binding was observed with LacY stabilized in an outward-open conformation (k(on) = 4-20 mu M-1.s(-1)), indicating unobstructed access to the binding site even for ligands that are much larger than lactose. Dissociation rate constants (k(off)) increase with the size of the aglycone so that K-d values also increase but remain in the micromolar range for each homolog. Phe27 (helix I) forms an apparent constriction in the pathway for sugar by protruding into the periplasmic cavity. However, replacement of Phe27 with a bulkier Trp does not create an obstacle in the pathway even for large ligands, since binding kinetics remain unchanged. High accessibility of the binding site is also observed in a LacY/nanobody complex with partially blocked periplasmic opening. Remarkably, E. coli expressing WT LacY catalyzes transport of alpha- or beta-galactopyranosides with oversized aglycones such as bodipy or Aldol518, which may require an extra space within the occluded intermediate. The results confirm that LacY specificity is strictly directed toward the galactopyranoside ring and also clearly indicate that the opening on the periplasmic side is sufficiently wide to accommodate the large galactoside derivatives tested here. We conclude that the actual pathway for the substrate entering from the periplasmic side is wider than the pore diameter calculated in the periplasmic-open X-ray structures.
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