Structural modeling of dual-affinity purified Pho84 phosphate transporter.
The phosphate transporter Pho84 of Saccharomyces cerevisiae is predicted to contain 12 transmembrane (TM) regions, divided into two partially duplicated parts of 6 TM segments. The three-dimensional (3D) organization of the Pho84 protein has not yet been determined. However, the 3D crystal structure of the Escherichia coli MFS glycerol-3-phosphate/phosphate antiporter, GlpT, and lactose transporter, LacY, has recently been determined. On the basis of extensive prediction and fold recognition analyses (at the MetaServer), GlpT was proposed as the best structural template on which the arrangement of TM segments of the Pho84 transporter was fit, using the comparative structural modeling program MODELLER. To initiate an evaluation of the appropriateness of the Pho84 model, we have performed two direct tests by targeting spin labels to putative TM segments 8 and 12. Electron paramagnetic resonance spectroscopy was then applied on purified and spin labeled Pho84. The line shape from labels located at both positions is consistent with the structural environment predicted by the template-generated model, thus supporting the model.
Lagerstedt
Jens O
JO
Department of Chemistry and Biomedical Sciences, Kalmar University, S-391 82 Kalmar, Sweden.
Voss
John C
JC
Wieslander
Ake
A
Persson
Bengt L
BL
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002846:Chromatography, Affinity; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D008958:Models, Molecular; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D028081:Proton-Phosphate Symporters / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013113:Spin Labels
D005656:Fungal Proteins; C105495:PHO84 protein, S cerevisiae; D028081:Proton-Phosphate Symporters; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D013113:Spin Labels