Unitat de Biofísica, Departament de Bioquímica i de Biologia Molecular, Facultat de Medicina, and Centre d'Estudis en Biofísica, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain.
Proc Natl Acad Sci U S A. 2010 Dec 21;107(51):22078-83. doi: 10.1073/pnas.1008649107. Epub 2010 Dec 6.
The melibiose carrier from Escherichia coli (MelB) couples the accumulation of the disaccharide melibiose to the downhill entry of H(+), Na(+), or Li(+). 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(+) binding. Secondly, though Asp124 is not essential for Na(+) binding, this acidic residue may play a critical role, possibly by its interaction with the bound cation, in the full Na(+)-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(+) 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.
大肠杆菌的棉子糖载体（MelB）将二糖棉子糖的积累与 H(+)、Na(+)或 Li(+)的下坡进入偶联。在这项工作中，使用底物诱导的傅里叶变换红外差谱法与荧光光谱法相结合，定量比较了先前涉及钠离子结合的 MelB 突变体与完全无半胱氨酸的 MelB 透酶的构象特性。结果首先表明 Asp55 和 Asp59 是 Na(+)结合的必需配体。其次，尽管 Asp124 对于 Na(+)结合不是必需的，但这个酸性残基可能通过与结合阳离子的相互作用在完全 Na(+)诱导的构象变化中发挥关键作用，这些构象变化对于离子和糖结合位点之间的有效偶联是必需的；该残基也可能是糖配体。第三，Asp19 不参与 Na(+)结合，但它是棉子糖的配体。这些残基在 MelB 的两个独立穿线模型中的位置与其提出的作用一致。
