Homology Model of the Na + /Proline Transporter PutP of Escherichia coli and Its Functional Implications Elena Olkhova 1 ⁎†, Michael Raba 2 †, Susanne Bracher 2 , Daniel Hilger 2 and Heinrich Jung 2 ⁎ 1 Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max-von-Laue Str. 3, D-60438 Frankfurt am Main, Germany 2 Mikrobiologie, Biozentrum der LMU München, Groβhaderner Strasse 2-4, 82152 Martinsried, Germany Received 7 July 2010; received in revised form 19 November 2010; accepted 22 November 2010 Available online 3 December 2010 Edited by J. Bowie Keywords: secondary transport; PutP; sodium/solute symport; molecular modeling; docking 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 conser- vation 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 computation- ally 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. © 2010 Elsevier Ltd. All rights reserved. Introduction Sodium solute symporters (SSS) form a large family of membrane transport proteins (TC 2A.21, SLC5) that co-transport Na + with sugars, amino acids, inorganic ions, and some vitamins. 1–3 Mem- bers of this family are important in human physi- ology. The function of several family members has been well characterized, including the mammalian glucose (SGLTs), the iodide (NIS), the Vibrio para- haemolyticus galactose/glucose (vSGLT), and the Escherichia coli proline (PutP) symporters. Like other secondary transporters, the SSS proteins are thought to accomplish transport via alternating access of the substrate binding site to either side of the membrane. 4,5 Experimental evidence for this mechanism comes from numerous biochemical analyses and more recently from X-ray crystallo- graphic data (e.g., Refs. 6,7). The Na + /proline transporter PutP of E. coli is a biochemically well-characterized prokaryotic mem- ber of the SSS family. 8,9 It catalyzes the coupled translocation of Na + and proline with a stoichiom- etry of 1:1. Kinetic analyses of Na + /solute symport catalyzed by different members of the SSS family *Corresponding authors. E-mail addresses: Elena.Olkhova@biophys.mpg.de; hjung@lmu.de. † E.O. and M.R. contributed equally to this work. Abbreviations used: FM, fluorescein-5-maleimide; MTSET, methanethiosulfonate ethyltrimethylammonium; NEM, N-ethylmaleimide; PutP(ΔCys), engineered transporter devoid of all five native cysteine residues; SSS family, Na + /solute symporter family (TC 2A.21, SLC5); TMS, putative transmembrane segment. doi:10.1016/j.jmb.2010.11.045 J. Mol. Biol. (2011) 406, 59–74 Contents lists available at www.sciencedirect.com Journal of Molecular Biology journal homepage: http://ees.elsevier.com.jmb 0022-2836/$ - see front matter © 2010 Elsevier Ltd. All rights reserved.