Role of the irreplaceable residues in the LacY alternating access mechanism.
Few side chains in the galactoside/H(+) symporter LacY (lactose permease of Escherichia coli) are irreplaceable for an alternating access mechanism in which sugar binding induces closing of the cytoplasmic cavity and reciprocal opening of a periplasmic cavity. In this study, each irreplaceable residue was mutated individually, and galactoside-induced opening or closing of periplasmic or cytoplasmic cavities was probed by site-directed alkylation. Mutation of Glu126 (helix IV) or Arg144 (helix V), which are essential for sugar binding, completely blocks sugar-induced periplasmic opening as expected. Remarkably, however, replacement of Glu269 (helix VIII), His322 (helix X), or Tyr236 (helix VII) causes spontaneous opening of the periplasmic cavity in the absence of sugar and decreased closing of the cytoplasmic cavity in the presence of galactoside. In contrast, mutation of Arg302 (helix IX) or Glu325 (helix X) has no such effect, and sugar binding induces normal opening and closing of periplasmic and cytoplasmic cavities. Possibly, Glu269, His322, and Tyr236 act in concert to coordinate opening and closing of the cavities by binding water, which also acts as a cofactor in H(+) translocation. Mutation of the triad results in loss of the bound water, which destabilizes LacY, and the cavities open and close in an uncoordinated manner. Thus, the triad mutants exhibit poor affinity for sugar, and galactoside/H(+) symport is abolished as well.
Department of Physiology, University of California, Los Angeles, CA 90095, USA.
Yonggang
Y
Zhou
Xiaoxu
X
Jiang
H Ronald
HR
Kaback
D019943:Amino Acid Substitution; D001692:Biological Transport / Q000502:physiology; D003593:Cytoplasm / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007785:Lactose / Q000235:genetics / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D019897:Periplasm / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D027981:Symporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D009004:Monosaccharide Transport Proteins; D027981:Symporters; D007785:Lactose