Abstract
Previous photolabeling and limited proteolysis studies suggested that one of the four basic residues (Arg-141) of the N-terminal cytoplasmic loop connecting helices IV and V (loop 4-5) of the melibiose permease (MelB) from Escherichia coli has a potential role in its symport function (Ambroise, Y., Leblanc, G., and Rousseau, B. (2000) Biochemistry 39, 1338-1345). A mutagenesis study of Arg-141 and of the other three basic residues of loop 4-5 was undertaken to further examine this hypothesis. Cys replacement analysis indicated that Arg-141 and Arg-149, but not Lys-138 and Arg-139, are essential for MelB transport activity. Replacement of Arg-141 by neutral residues (Cys or Gln) inactivated transport and energy-independent carrier-mediated flows of substrates (counterflow, efflux), whereas it had a limited effect on co-substrate binding. R141C sugar transport was partially rescued on reintroducing a positive charge with a charged and permeant thiol reagent. Whereas R149C was completely inactive, R149K and R149Q remained functional. Strikingly, introduction of an additional mutation in the C-terminal helix X (Gly for Val-343) of R149C restored sugar transport. Impermeant thiol reagents inhibited R149C/V343G transport activity in right-side-out membrane vesicles and prevented sugar binding in a sugar-protected manner. All these data suggest that MelB loop 4-5 is close to the sugar binding site and that the charged residue Arg-141 is involved in the reaction of co-substrate translocation or substrate release in the inner compartment.
Highlights
Ose, raffinose) or -galactosides (methyl-1-thio--galactopyranoside (TMG)) using a favorable electrochemical potential gradient for Naϩ, Liϩ, or Hϩ [1,2,3]
To elucidate the role of Arg-141 and loop 4 –5, we analyzed the functional consequences of replacing Arg-141 by different residues (Cys, Lys, Gln) and compared them with the effects produced by the mutagenesis of the three other basic residues of this loop (Lys-138, Arg-139, and Arg-149)
The obtained data suggest that the cytoplasmic loop 4 –5 of melibiose permease (MelB) is close to the sugar binding site and may participate directly in co-substrate translocation by MelB
Summary
MelB, melibiose permease, (6-O-␣-Dgalactopyranosyl-D-glucose); TMG, methyl-1-thio--galactopyranoside; ␣-NPG, p-nitrophenyl ␣-D-6-galactopyranoside; FRET, fluorescence resonance energy transfer; Dns2-S-Gal, 2Ј-(N-dansyl)aminoethyl-1-thio-D-galactopyranoside; MTSEAϩ, (2-aminoethyl)methanethiosulfonate hydrobromide; MTSETϩ, (2-(trimethylammonium)ethyl) methanethiosulfonate hydrobromide; PCMBSϪ, p-chloromercury benzoic acid; RSO membranes, right-side-out membranes. Biochemical, and spectroscopic approaches have been extensively used to collect information on the functional organization of the transporter This organization involves the presence of a potential coordination network for the cation recognition and coupling mechanism, including four aspartic acid residues distributed in the inner halves of helices I (Asp-19), II (Asp-55 and Asp-59), and IV (Asp-124) of the N-terminal hydrophobic domain of MelB [6, 15,16,17,18]. Extensive Cys-scanning mutagenesis of residues of membrane domains combined with chemical modifications of MelB with impermeant thiol reagents or with second-site mutation analysis has been used to identify sectors of given membrane domains facing the co-substrate pathway They suggest proximity between helices I, II, IV, VII, X, and XI (see Ref. 20 and references therein). The obtained data suggest that the cytoplasmic loop 4 –5 of MelB is close to the sugar binding site and may participate directly in co-substrate translocation by MelB
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