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

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

doi:10.1074/jbc.m601259200

Cited by 43 publications

(52 citation statements)

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“…In these experiments the sugar was added at a concentration of 50 mM (Fig. S2), a value close to the half-saturating concentration of C-less for melibiose (30). Because protons are present in the medium, substrate translocation becomes possible and it may contribute also to the difference spectra.…”

Section: Resultsmentioning

confidence: 99%

“…Accordingly, the MelB fold most likely consists in two distinct N-terminal and a C-terminal six-helix bundles, pseudosymmetrically related by an axis running through a central cavity nearly perpendicular to the membrane (24), as other members of the MFS superfamily show (28). As for the above cited symporters, the MelB transport cycling model includes several intermediate steps (12,30) (Fig. S1).…”

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confidence: 99%

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

Granell

León²,

Leblanc³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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

Granell

León²,

Leblanc³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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“…The previous spectroscopic experiments were carried out on proteoliposomes in which MelB has an orientation opposite to that in the cell (4,39). The absence of binding of the mutated protein when reconstituted in proteoliposomes could be due to a conformation locked open toward the inward-facing space, as reported previously for R149C (39), or to an irreversible denaturizing effect of detergents during protein purification (although the spectral comparison in Fig.…”

Section: Defective Binding Of Substrates To Lys-377 Mutants: Infraredmentioning

confidence: 93%

“…It is of help to consider other mutations involving Ile-22. Therefore, I22S has been described as partly compensating the loss of the negative charge in E142C (4), and mutations at Ile-22 have been found as second-site revertants for A350C, A383C, L391C, and G395C (8,51). Given the diversity of locations of these side chains, it seems evident that further experiments are needed to establish an unequivocal role for Ile-22.…”

Section: Discussionmentioning

confidence: 99%

“…Cotransport with Na ϩ or Li ϩ involves first cation binding, which increases the melibiose affinity. This is followed by the binding of sugar, conformational changes leading to an inward-open conformation, release of sugar, and, finally, release of the cation (1,3,4). Some important residues for the transport mechanism, especially for the binding step, have been identified.…”

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The Melibiose Transporter of Escherichia coli

Fuerst

Lin

Granell

et al. 2015

Journal of Biological Chemistry

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Studying Substrate Binding to Reconstituted Secondary Transporters by Attenuated Total Reflection Infrared Difference Spectroscopy

Lórenz-Fonfrı́a¹,

León²,

Padrós³

2012

Methods in Molecular Biology

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The determination of protein conformational changes induced by the interaction of substrates with secondary transporters is an important step toward the elucidation of their transport mechanism. Since conformational changes in a protein alter its vibrational patterns, they can be detected with high sensitivity by infrared difference (IR(diff)) spectroscopy without the need for external probes. We describe a general procedure to obtain substrate-induced IR(diff) spectra by alternating perfusion of buffers over an attenuated total reflection (ATR) crystal containing an adhered film of a membrane protein reconstituted in lipids. As an example, we provide specific protocols to obtain melibiose and Na(+)-induced ATR-IR(diff) spectra of reconstituted melibiose permease, a sodium/melibiose co-transporter from E. coli. The presented methodology is applicable in principle to any membrane protein, provided that it can be purified and reconstituted in functional form, and appropriate substrates are available.

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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

Cited by 43 publications

References 53 publications

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

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

The Melibiose Transporter of Escherichia coli

Studying Substrate Binding to Reconstituted Secondary Transporters by Attenuated Total Reflection Infrared Difference Spectroscopy

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