Structural insights into the activation mechanism of melibiose permease by sodium binding

Abstract

The melibiose carrier from Escherichia coli (MelB) couples the accumulation of the disaccharide melibiose to the downhill entry of H þ , Na þ , or Li þ . In this work, substrate-induced FTIR difference spectroscopy was used in combination with fluorescence spectroscopy to quantitatively compare the conformational properties of MelB mutants, implicated previously in sodium binding, with those of a fully functional Cys-less MelB permease. The results first suggest that Asp55 and Asp59 are essential ligands for Na þ binding. Secondly, though Asp124 is not essential for Na þ binding, this acidic residue may play a critical role, possibly by its interaction with the bound cation, in the full Na þ -induced conformational changes required for efficient coupling between the ion-and sugar-binding sites; this residue may also be a sugar ligand. Thirdly, Asp19 does not participate in Na þ binding but it is a melibiose ligand. The location of these residues in two independent threading models of MelB is consistent with their proposed role.infrared spectroscopy | ligand binding | membrane proteins | sugar/cation symporter A ccording to the chemiosmotic principles, secondary active transporters comprise membrane proteins that couple in an obligatory fashion the discharge of an ionic gradient (or that of a solute gradient) to the "uphill" translocation of different solutes in the same direction (symporters or cotransporters) or in the opposite one (antiporters or exchangers) (1). Thermodynamic considerations and a wealth of kinetic, biochemical, and biophysical studies have led to the consensual view that substrate translocation relies on the alternating-access concept (2), stating that at any moment a single binding site in a polar cavity is accessible to only one side of the membrane (see for example, recent reviews and references therein in refs. 3-8). The recent elucidation of the atomic structure of almost a dozen of transporters provides strong support to the validity of the alternatingaccess concept (see reviews cited above). Finally, the diversity of conformation(s) adopted in the different transporters crystals has yielded insights into the structural basis of the various steps of the symporter cycle. Still, many issues regarding the conformational changes, especially those involved in ligand binding and in the coupling of the ligand binding sites, remain largely unanswered.In this context, the melibiose permease (MelB) of Escherichia coli, which belongs to the Glycoside-Pentoside-Hexuronide: Cation symporter family (9) (a submember of the major facilitator superfamily, MFS), is a convenient Na þ symporter to analyze the molecular and structural basis of the interaction of the coupling ion with the transporter. MelB efficiently couples the uphill transport of α-or β-galactosides to the favorable entry of Na þ , Li þ , or H þ (H 3 O þ ) (10,11). In the past, this property has been extensively exploited to investigate the molecular and structural basis of the ion-MelB interaction and implications in the coupling propert...

The Melibiose Transporter of Escherichia coli

“…Tyr-120 (HIV) appears to separate the two areas. Being located between the two binding sites, Asp-124 most probably can orient indistinctly toward the cationic or the sugar site, giving rise to conformational changes upon cation binding that increase the sugar affinity, as proposed previously (5,6). The MD simulations run after placing a Na ϩ in the Asp-55/Asp-59 environment clearly give credit to this presumption because, in this case, Asp-124 moves toward the Asp-55/Asp-59 site.…”

“…In a previous work, we studied mutants of these side chains and showed that Asp-55 and Asp-59 are essential ligands for Na ϩ (5). Regarding the role of Asp-124, we showed that it is an essential residue for sugar binding, required as well for normal Na ϩ binding and Na more, a close proximity between Asp-55 and Asp-59 is probably required for Na ϩ binding because we know that mutants of Asp-55 or Asp-59 alone lack Na ϩ binding (5,(43)(44)(45)(46). We therefore suggest that the main role of Lys-377 is to stabilize the inner region of MelB by compensating repulsive interactions between the anionic side chains of Asp-55 and Asp-59.…”

“…Furthermore, a conformational change involving Asp-124 has been suggested to follow Na ϩ binding (5,6). To find out the effect of Na ϩ binding on the Lys-377 (HXI) and the Asp-55/ Asp-59 dyad (HII), we manually inserted an Na ϩ in place of a water molecule located between the two Asp side chains and performed MD simulations as above.…”

“…According to the crystallographic structure (6), Lys-377 is located in the inner space of the protein near Asp-55 and Asp-59 ( Fig. 1), side chains that have been shown to be important for substrate binding (5). Furthermore, Lys-377 has been proposed to interact with Asp-59 (8).…”

“…Some important residues for the transport mechanism, especially for the binding step, have been identified. Therefore, it is believed that the cation binding site comprises at least Asp-55, Asp-59 (helix II), and Asp-124 (helix IV), whereas the sugar most likely interacts at least with Asp-19 and Asp-124 (helices I and IV) (2,5,6).…”