title: Role of Conserved Gly-Gly Pairs on the Periplasmic Side of LacY.
abstract: On the periplasmic side of LacY, two conserved Gly-Gly pairs in helices II and XI (Gly46 and Gly370, respectively) and helices V and VIII (Gly159 and Gly262, respectively) allow close packing of each helix pair in the outward (periplasmic)-closed conformation. Previous studies demonstrate that replacing one Gly residue in each Gly-Gly pair with Trp leads to opening of the periplasmic cavity with abrogation of transport activity, but an increased rate of galactoside binding. To further investigate the role of the Gly-Gly pairs, 11 double-replacement mutants were constructed for each pair at positions 46 (helix II) and 262 (helix VIII). Replacement with Ala or Ser results in decreased but significant transport activity, while replacements with Thr, Val, Leu, Asn, Gln, Tyr, Trp, Glu, or Lys exhibit very little or no transport. Remarkably, however, the double mutants bind galactoside with affinities 10-20-fold higher than that of the pseudo-WT or WT LacY. Moreover, site-directed alkylation of a periplasmic Cys replacement indicates that the periplasmic cavity becomes readily accessible in the double-replacement mutants. Molecular dynamics simulations with the WT and double-Leu mutant in the inward-open/outward-closed conformation provide support for this interpretation.
authors.forename: Xiaoxu
authors.initials: X
authors.lastname: Jiang
authors.affiliation: Department of Theoretical Physics and Swedish e-Science Research Center, Science for Life Laboratory, KTH Royal Institute of Technology , SE-171 21 Solna, Sweden.
authors.forename: Magnus
authors.initials: M
authors.lastname: Andersson
authors.forename: Bryan T
authors.initials: BT
authors.lastname: Chau
authors.forename: Larissa Y
authors.initials: LY
authors.lastname: Wong
authors.forename: Maria Katerina R
authors.initials: MK
authors.lastname: Villafuerte
authors.forename: H Ronald
authors.initials: HR
authors.lastname: Kaback
mesh_terms: D000478:Alkylation; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001693:Biological Transport, Active; D017124:Conserved Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006033:Glycylglycine / Q000737:chemistry / Q000235:genetics; D007785:Lactose / Q000378:metabolism; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D009004:Monosaccharide Transport Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009598:Nitrophenylgalactosides / Q000378:metabolism; D019897:Periplasm / Q000378:metabolism; D011487:Protein Conformation; D000072756:Protein Conformation, alpha-Helical; D027981:Symporters / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D009004:Monosaccharide Transport Proteins; D027981:Symporters; D006033:Glycylglycine; D009598:Nitrophenylgalactosides; C059200:4-nitrophenylgalactoside; D007785:Lactose