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.
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
You can help EcoliWiki by summarizing why this paper is useful
Useful Materials and Methods
You can help Ecoliwiki by describing the useful materials (strains, plasmids, antibodies, etc) described in this paper.
|Gene product||Qualifier||GO ID||GO term name||Evidence Code||with/from||Aspect||Notes||Status|
Add links to pages that link here (e.g. gene, product, method pages)
See Help:References for how to manage references in EcoliWiki.