| Abstract
| - Structural classification of families of membrane proteins by bioinformatics techniques hasbecome a critical aspect of membrane protein research. We have proposed hydropathy profile alignmentto identify structural homology between families of membrane proteins. Here, we demonstrateexperimentally that two families of secondary transporters, the ESS and 2HCT families, indeed sharesimilar folds. Members of the two families show highly similar hydropathy profiles but cannot be shownto be homologous by sequence similarity. A structural model was predicted for the ESS family transportersbased upon an existing model of the 2HCT family transporters. In the model, the transporters fold intotwo domains containing five transmembrane segments and a reentrant or pore-loop each. The two pore-loops enter the membrane embedded part of the proteins from opposite sides of the membrane. The modelwas verified by accessibility studies of cysteine residues in single-Cys mutants of the Na+-glutamatetransporter GltS of Escherichia coli, a member of the ESS family. Cysteine residues positioned in predictedperiplasmic loops were accessible from the periplasm by a bulky, membrane-impermeable thiol reagent,while cysteine residues in cytoplasmic loops were not. Furthermore, two cysteine residues in the predictedpore-loop entering the membrane from the cytoplasmic side were shown to be accessible for small,membrane-impermeable thiol reagents from the periplasm, as was demonstrated before for the Na+-citrate transporter CitS of Klebsiella pneumoniae, a member of the 2HCT family. The data strongly suggeststhat GltS of the ESS family and CitS of the 2HCT family share the same fold as was predicted by comparingthe averaged hydropathy profiles of the two families.
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