A structural model for the osmosensor, transporter, and osmoregulator ProP of Escherichia coli.
Transporter ProP of Escherichia coli, a member of the major facilitator superfamily (MFS), acts as an osmosensor and an osmoregulator in cells and after purification and reconstitution in proteoliposomes. H(+)-osmoprotectant symport via ProP is activated when medium osmolality is elevated with membrane impermeant osmolytes. The three-dimensional structure of ProP was modeled with the crystal structure of MFS member GlpT as a template. This GlpT structure represents the inward (or cytoplasm)-facing conformation predicted by the alternating access model for transport. LacZ-PhoA fusion analysis and site-directed fluorescence labeling substantiated the membrane topology and orientation predicted by this model and most hydropathy analyses. The model predicts the presence of a proton pathway within the N-terminal six-helix bundle of ProP (as opposed to the corresponding pathway found within the C-terminal helix bundle of its paralogue, LacY). Replacement of residues within the N-terminal helix bundle impaired the osmotic activation of ProP, providing the first indication that residues outside the C-terminal domain are involved in osmosensing. Some residues that were accessible from the periplasmic side, as predicted by the structural model, were more susceptible to covalent labeling in permeabilized membrane fractions than in intact bacteria. These residues may be accessible from the cytoplasmic side in structures not represented by our current model, or their limited exposure in vivo may reflect constraints on transporter structure that are related to its osmosensory mechanism.
Wood
Janet M
JM
Department of Molecular and Cellular Biology and Guelph-Waterloo Centre for Graduate Work in Chemistry and Biochemistry, University of Guelph, Guelph N1G 2W1, Ontario, Canada. jwood@uoguelph.ca
Culham
Doreen E
DE
Hillar
Alexander
A
Vernikovska
Yaroslava I
YI
Liu
Feng
F
Boggs
Joan M
JM
Keates
Robert A B
RA
D000469:Alkaline Phosphatase; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D001693:Biological Transport, Active; D018844:Cyclin-Dependent Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005456:Fluorescent Dyes; D007763:Lac Operon; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D027981:Symporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014882:Water-Electrolyte Balance; D001616:beta-Galactosidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; C079244:ProP protein, E coli; D011993:Recombinant Fusion Proteins; D027981:Symporters; D018844:Cyclin-Dependent Kinases; D000469:Alkaline Phosphatase; C114089:phoA protein, E coli; D001616:beta-Galactosidase