Clogging the periplasmic pathway in LacY.
The lactose permease of Escherichia coli (LacY) is a highly dynamic membrane transport protein. Crystal structures of wild-type and mutant LacY all exhibit an inward-facing conformation with an open cytoplasmic pathway and a tightly packed periplasmic side, which makes the binding site inaccessible from the outside. However, biochemical and biophysical findings provide strong evidence that occupation of the sugar-binding site leads to an increased probability of opening of a hydrophilic pathway on the periplasmic side and closing of the cytoplasmic cavity. By this means, the sugar-binding site becomes accessible to either side of the membrane in alternating fashion. To extend studies on the relationship between the periplasmic pathway and transport activity, engineered single-Cys replacements in the periplasmic pathway were reacted to completion with thiol reagents, and the effects on transport and sugar binding were tested. Inactivation correlates for the most part with the size of the modifying reagent, although the position of the Cys replacement is also important. However, sugar binding is unaffected. The results suggest that placement of a relatively large moiety in the putative periplasmic cleft of LacY likely prevents closure, an essential step in the transport cycle, without significantly altering access of sugar to the binding site.
Nie
Yiling
Y
Department of Physiology, Molecular Biology Institute, University of California, Los Angeles, California 90095, USA.
Zhou
Yonggang
Y
Kaback
H Ronald
HR
D018360:Crystallography, X-Ray; D003545:Cysteine / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019897:Periplasm / Q000737:chemistry / Q000378:metabolism*; D017433:Protein Structure, Secondary / Q000235:genetics; D012235:Rhodamines / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics; D027981:Symporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D012235:Rhodamines; D027981:Symporters; C005358:tetramethylrhodamine; C021499:lactose permease; D003545:Cysteine