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J. Biol. Chem., Vol. 283, Issue 8, 4921-4929, February 22, 2008

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Role of Ser-340 and Thr-341 in Transmembrane Domain IX of the Na⁺/Proline Transporter PutP of Escherichia coli in Ligand Binding and Transport^*

Daniel Hilger, Maret Böhm^¶1, Alexandra Hackmann^¶2, and Heinrich Jung^¶3

From the LMU Munich, Department Biology I, Microbiology and Munich Center for integrated Protein Science (CiPS^M), D-80638 Munich, Germany and ^¶University of Osnabrück, Department of Microbiology, D-49069 Osnabrück, Germany

The Na⁺/solute symporter family comprises more than 400 members of pro- and eukaryotic origin. Using the Na⁺/proline transporter PutP of Escherichia coli as a model, the role of two conserved residues, Ser-340 and Thr-341, is investigated to obtain insights into the mechanism of transport catalyzed by members of this family. Substitution of these amino acids alters the transport kinetics of cells and proteoliposomes containing the PutP variants significantly. In particular, the apparent affinities for Na⁺ and Li⁺ are reduced by 2 orders of magnitude or more. Also proline binding is affected, albeit to a lesser extent than ion binding. Thereby, the presence of a hydroxyl group at position 341 is essential for high affinity ligand binding. Furthermore, Cys placed at position 340 or 341 reacts with sulfhydryl reagents of different polarity, indicating accessibility from the water phase. In addition, Cys cross-linking suggests proximity of the residues to other amino acids previously shown to be crucial for ligand binding. For these reasons it is suggested that Ser-340 and Thr-341 are located in a ligand translocation pathway. Furthermore, it is proposed that the side chain of Thr-341 directly participates in Na⁺ binding.

Received for publication, August 14, 2007 , and in revised form, December 11, 2007.

^* This work was financially supported by the Deutsche Forschungsgemeinschaft Grants Ju333/3-2, Ju333/4-2, and Exc114-1). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental material including Table 1 and Figs. 1, 2, 3, 4.

¹ Current address: Cancer Research Program, Garvan Institute of Medical Research, 384 Victoria St, Darlinghurst, 2010 NSW, Sydney, Australia.

² Current address: Institute of Molecular Biology and Tumor Research, Philipps-University Marburg, Emil Mannkopff-Strasse 2, 35032 Marburg, Germany.

³ To whom correspondence should be addressed: LMU Munich, Dept. Biology I, Microbiology, Maria-Ward-Strasse 1a, D-80638 Munich, Germany. Fax: 49-89-2180-63857; E-mail: hjung{at}lmu.de.

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