A 3D structure model of the melibiose permease of Escherichia coli represents a distinctive fold for Na+ symporters

Department of Cell Physiology and Molecular Biophysics, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.81). 09/2009; 106(36):15291-6. DOI: 10.1073/pnas.0905516106
Source: PubMed

ABSTRACT The melibiose permease of Escherichia coli (MelB) catalyzes the coupled stoichiometric symport of a galactoside with a cation (either Na(+), Li(+), or H(+)), using free energy from the downhill translocation of one cosubstrate to catalyze the accumulation of the other. Here, we present a 3D structure model of MelB threaded through a crystal structure of the lactose permease of E. coli (LacY), manually adjusted, and energetically minimized. The model contains 442 consecutive residues ( approximately 94% of the polypeptide), including all 12 transmembrane helices and connecting loops, with no steric clashes and superimposes well with the template structure. The electrostatic surface potential calculated from the model is typical for a membrane protein and exhibits a characteristic ring of positive charges around the periphery of the cytoplasmic side. The 3D model indicates that MelB consists of two pseudosymmetrical 6-helix bundles lining an internal hydrophilic cavity, which faces the cytoplasmic side of the membrane. Both sugar and cation binding sites are proposed to lie within the internal cavity. The model is consistent with numerous previous mutational, biochemical/biophysical characterizations as well as low-resolution structural data. Thus, an alternating access mechanism with sequential binding is discussed. The proposed overall fold of MelB is different from the available crystal structures of other Na(+)-coupled transporters, suggesting a distinctive fold for Na(+) symporters.

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    ABSTRACT: In a variety of bacteria, the phosphotransferase protein IIAGlc plays a key regulatory role in catabolite repression in addition to its role in the vectorial phosphorylation of glucose catalyzed by the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). The lactose permease (LacY) of Escherichia coli catalyzes stoichiometric symport of a galactoside with an H+, using a mechanism in which sugar- and H+-binding sites become alternatively accessible to either side of the membrane. Both the expression (via regulation of cAMP levels) and the activity of LacY are subject to regulation by IIAGlc (inducer exclusion). Here we report the thermodynamic features of the IIAGlc-LacY interaction as measured by isothermal titration calorimetry (ITC). The studies show that IIAGlc binds to LacY with a Kd of about 5 muM and a stoichiometry of unity and that binding is driven by solvation entropy and opposed by enthalpy. Upon IIAGlc binding, the conformational entropy of LacY is restrained, which leads to a significant decrease in sugar affinity. By suppressing conformational dynamics, IIAGlc blocks inducer entry into cells and favors constitutive glucose uptake and utilization. Furthermore, the studies support the notion that sugar binding involves an induced-fit mechanism that is inhibited by IIAGlc binding. The precise mechanism of the inhibition of LacY by IIAGlc elucidated by ITC differs from the inhibition of melibiose permease (MelB), supporting the idea that permeases can differ in their thermodynamic response to binding IIAGlc.
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    ABSTRACT: The phosphotransfer protein IIA(Glc) of the bacterial phosphoenolpyruvate:carbohydrate phosphotransferase system plays a key role in the regulation of carbohydrate metabolism. Melibiose permease (MelB) is one among several permeases subject to IIA(Glc) regulation. The regulatory mechanisms are poorly understood; in addition, thermodynamic features of IIA(Glc) binding to other proteins are also unknown. Applying isothermal titration calorimetry (ITC) and amine-specific cross-linking, we show that IIA(Glc) directly binds to MelB of Salmonella typhimurium (MelBSt) and Escherichia coli MelB (MelBEc) at a stoichiometry of unity in the absence or presence of melibiose. The dissociation constant values are 3-10 μM for MelBSt and 25 μM for MelBEc. All of the binding is solely driven by favorable enthalpy forces. IIA(Glc) binding to MelBSt in the absence or presence of melibiose yields a large negative heat capacity change; in addition, the conformational entropy is constrained upon the binding. We further found that the IIA(Glc)-bound MelBSt exhibits a decreased binding-affinity for melibiose or nitrophenyl-α-galactoside. It is believed that sugar binding to the permease involves in an induced-fit mechanism, and the transport process requires conformational cycling between different states. Thus, the thermodynamic data are consistent with the interpretation that IIA(Glc) inhibits the induced-fit process and restricts the conformational dynamics of MelBSt.
    Journal of Biological Chemistry 10/2014; 290(11). DOI:10.1074/jbc.M114.609255 · 4.60 Impact Factor

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