Homology model of the Na+/proline transporter PutP of Escherichia coli and its functional implications.
Na(+)/solute symporters are essential membrane integrated proteins that couple the flow of Na(+) ions driven by electrochemical Na(+) gradients to the transport of solutes across biological membranes. Here, we used a combination of molecular modeling techniques and evolutionary conservation analysis to construct and validate a first model of the Na(+)/proline symporter PutP of Escherichia coli based on the crystal structure of the bacterial Na(+)/galactose symporter vSGLT. Ligand docking experiments were employed to gain information about residues involved in proline binding. The proposed model is consistent with the available experimental data and was further validated by amino acid substitutions and kinetic and protein chemical analyses. Combination of the results of molecular modeling and functional studies predicts the location and organization of the Na(+) and proline binding sites. Remarkably, as proposed computationally and discovered here experimentally, residues Y140, W244, and Y248 of transmembrane segments 4 and 7 are found to be particularly important for PutP function and suggested to participate in proline binding and/or gating.
Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max-von-Laue Str. 3, D-60438 Frankfurt am Main, Germany. Elena.Olkhova@biophys.mpg.de
Elena
E
Olkhova
Michael
M
Raba
Susanne
S
Bracher
Daniel
D
Hilger
Heinrich
H
Jung
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D026921:Amino Acid Transport Systems, Neutral / Q000737:chemistry*; D001665:Binding Sites; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D005690:Galactose / Q000737:chemistry; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D040681:Structural Homology, Protein; D027981:Symporters / Q000737:chemistry*
D026921:Amino Acid Transport Systems, Neutral; D029968:Escherichia coli Proteins; D027981:Symporters; C060609:PutP protein, E coli; D005690:Galactose