title: Trp replacements for tightly interacting Gly-Gly pairs in LacY stabilize an outward-facing conformation.
abstract: Trp replacements for conserved Gly-Gly pairs between the N- and C-terminal six-helix bundles on the periplasmic side of lactose permease (LacY) cause complete loss of transport activity with little or no effect on sugar binding. Moreover, the detergent-solubilized mutants exhibit much greater thermal stability than WT LacY. A Cys replacement for Asn245, which is inaccessible/unreactive in WT LacY, alkylates readily in the Gly→Trp mutants, indicating that the periplasmic cavity is patent. Stopped-flow kinetic measurements of sugar binding with the Gly→Trp mutants in detergent reveal linear dependence of binding rates on sugar concentration, as observed with WT or the C154G mutant of LacY, and are compatible with free access to the sugar-binding site in the middle of the molecule. Remarkably, after reconstitution of the Gly→Trp mutants into proteoliposomes, the concentration dependence of sugar-binding rates increases sharply with even faster rates than measured in detergent. Such behavior is strikingly different from that observed for reconstituted WT LacY, in which sugar-binding rates are independent of sugar concentration because opening of the periplasmic cavity is limiting for sugar binding. The observations clearly indicate that Gly→Trp replacements, which introduce bulky residues into tight Gly-Gly interdomain interactions on the periplasmic side of LacY, prevent closure of the periplasmic cavity and, as a result, shift the distribution of LacY toward an outward-open conformation.
authors.affiliation: Department of Physiology, University of California, Los Angeles, CA 90095, USA.
authors.forename: Irina
authors.initials: I
authors.lastname: Smirnova
authors.forename: Vladimir
authors.initials: V
authors.lastname: Kasho
authors.forename: Junichi
authors.initials: J
authors.lastname: Sugihara
authors.forename: H Ronald
authors.initials: HR
authors.lastname: Kaback
mesh_terms: D001693:Biological Transport, Active / Q000502:physiology; D004926:Escherichia coli / Q000201:enzymology*; D005998:Glycine / Q000737:chemistry*; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008958:Models, Molecular; D016296:Mutagenesis; D009841:Oligonucleotides / Q000235:genetics; D011487:Protein Conformation; D011510:Proteolipids / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry*
chemical_list: D026901:Membrane Transport Proteins; D009841:Oligonucleotides; D011510:Proteolipids; C020485:proteoliposomes; D014364:Tryptophan; C021499:lactose permease; D005998:Glycine
keywords: alternating access; fluorescence; major facilitator superfamily; permease; symport