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J. Biol. Chem., Vol. 281, Issue 36, 25882-25892, September 8, 2006

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The Inner Interhelix Loop 4–5 of the Melibiose Permease from Escherichia coli Takes Part in Conformational Changes after Sugar Binding^*

Kerstin Meyer-Lipp¹, Natacha Séry, Constanta Ganea^¶2, Cécile Basquin^||, Klaus Fendler³, and Gérard Leblanc

From the Max Planck Institute of Biophysics, Max-von-Laue-Strasse 3, 60438 Frankfurt/M, Germany, the Université de Nice Sophia-Antipolis, the Commissariat à l'Énergie Atomique (LRC-CEA 16V), 06230 Villefranche sur mer, France, the ^¶Department of Biophysics, Carol Davila Medical University, 050474 Bucharest, Romania, and the ^||Unité TIRO, CEA, Université de Nice Sophia-Antipolis, Faculté de Médecine, 06107 Nice, France

Cytoplasmic loop 4–5 of the melibiose permease from Escherichia coli is essential for the process of Na⁺-sugar translocation (Abdel-Dayem, M., Basquin, C., Pourcher, T., Cordat, E., and Leblanc, G. (2003) J. Biol. Chem. 278, 1518–1524). In the present report, we analyze functional consequences of mutating each of the three acidic amino acids in this loop into cysteines. Among the mutants, only the E142C substitution impairs selectively Na⁺-sugar translocation. Because R141C has a similar defect, we investigated these two mutants in more detail. Liposomes containing purified mutated melibiose permease were adsorbed onto a solid supported lipid membrane, and transient electrical currents resulting from different substrate concentration jumps were recorded. The currents evoked by a melibiose concentration jump in the presence of Na⁺, previously assigned to an electrogenic conformational transition (Meyer-Lipp, K., Ganea, C., Pourcher, T., Leblanc, G., and Fendler, K. (2004) Biochemistry 43, 12606–12613), were much smaller for the two mutants than the corresponding signals in cysteineless MelB. Furthermore, in R141C the stimulating effect of melibiose on Na⁺ affinity was lost. Finally, whereas tryptophan fluorescence spectroscopy revealed impaired conformational changes upon melibiose binding in the mutants, fluorescence resonance energy transfer measurements indicated that the mutants still show cooperative modification of their sugar binding sites by Na⁺. These data suggest that: 1) loop 4–5 contributes to the coordinated interactions between the ion and sugar binding sites; 2) it participates in an electrogenic conformational transition after melibiose binding that is essential for the subsequent obligatory coupled translocation of substrates. A two-step mechanism for substrate translocation in the melibiose permease is suggested.

Received for publication, February 8, 2006 , and in revised form, June 20, 2006.

^* This work was supported in part by a grant from the Commissariat à l'Énergie Atomique. 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.

¹ Supported by a travel allowance from the Boehringer Ingelheim Fonds, a scholarship from the German Academic Exchange Service (Doktorandenstipendium im Rahmen des gemeinsamen Hochschulsonderprogramms III von Bund und Ländern), and the Max Planck Society.

² Recipient of a short-term Max Planck Society fellowship and of partial support from the Ministry of Education, Romania.

³ To whom correspondence should be addressed. Tel.: 49-69-6303-2035; Fax: 49-69-6303-2002; E-mail: klaus.fendler{at}mpibp-frankfurt.mpg.de.

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