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Zhou, Y, Nie, Y and Kaback, HR (2009) Residues gating the periplasmic pathway of LacY. J. Mol. Biol. 394:219-25


X-ray crystal structures of LacY (lactose permease of Escherichia coli) exhibit a large cytoplasmic cavity containing the residues involved in sugar binding and H(+) translocation at the apex and a tightly packed side facing the periplasm. However, biochemical and biophysical evidence provide a strong indication that a hydrophilic pathway opens on the external surface of LacY with closing of the cytoplasmic side upon sugar binding. Thus, an alternating-access mechanism in which sugar- and H(+)-binding sites at the approximate middle of the molecule are alternatively exposed to either side of the membrane is likely to underlie LacY-catalyzed sugar/H(+) symport. To further investigate periplasmic opening, we replaced paired residues on the tightly packed periplasmic side of LacY with Cys, and the effect of cross-linking was studied by testing the accessibility/reactivity of Cys148 with the elongated ( approximately 29 A), impermeant hydrophilic reagent maleimide-PEG2-biotin. When the paired-Cys mutant Ile40-->Cys/Asn245-->Cys containing native Cys148 is oxidized to form a disulfide bond, the reactivity of Cys148 is markedly inhibited. Moreover, the reactivity of Cys148 in this mutant increases with the length of the cross-linking agent. In contrast, maleimide-PEG2-biotin reactivity of Cys148 is unaffected by oxidation of two other paired-Cys mutants at the mouth of the periplasmic cavity. The data indicate that residues Ile40 and Asn245 play a primary role in gating the periplasmic cavity and provide further support for the alternating-access model.


PubMed PMC2784193 Online version:10.1016/j.jmb.2009.09.043


Catalysis; Cross-Linking Reagents/chemistry; Crystallography, X-Ray; Cysteine/chemistry; Cysteine/genetics; Cysteine/metabolism; Escherichia coli/enzymology; Escherichia coli/metabolism; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism; Lactose/metabolism; Monosaccharide Transport Proteins/chemistry; Monosaccharide Transport Proteins/genetics; Monosaccharide Transport Proteins/metabolism; Mutation; Periplasm/enzymology; Protein Conformation; Symporters/chemistry; Symporters/genetics; Symporters/metabolism


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