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

Kerstin Meyer-Lipp,Natacha Séry,Constanta Ganea,Cécile Basquin,Klaus Fendler,Gérard Leblanc

doi:10.1074/jbc.m601259200

Abstract

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.

Highlights

Consisting of 473 amino acids, this highly hydrophobic protein (70% apolar) has 12 transmembrane-spanning ␣-helices, organized as two asymmetric domains, each containing 6 helices, with its N and C termini facing the cytoplasm of the bacterium (6 –9)
In combination with spectroscopic evidence, the results suggest that loop 4 –5 contributes to the coordinated interactions between the ion and sugar binding sites and participates in conformational changes after melibiose binding
Initial rate and sugar accumulation at equilibrium in the presence of Naϩ (10 min) by D137C or E140C cells were 0.9 or 0.45 times the values measured in Cys-less melibiose permease (MelB), the level of E142C sugar accumulation was reduced by a factor of 10 (Fig. 1A)

Summary

Introduction

Consisting of 473 amino acids, this highly hydrophobic protein (70% apolar) has 12 transmembrane-spanning ␣-helices, organized as two asymmetric domains, each containing 6 helices, with its N and C termini facing the cytoplasm of the bacterium (6 –9). Functional Properties of D137C, E140C, and E142—Initial characterization of D137C, E140C, and E142C MelB mutants included: 1) the measurement of the time course of melibiose accumulation in bacteria in the presence or absence of saturating concentrations of activating Naϩ or Liϩ (10 mM) and 2) the estimation of the expression level of the mutated transporter.

Results

Conclusion