Role of Ser-340 and Thr-341 in transmembrane domain IX of the Na+/Proline transporter PutP of Escherichia coli in ligand binding and transport
Role of Ser-340 and Thr-341 in transmembrane domain IX of the Na+/Proline transporter PutP of Escherichia coli in ligand binding and transport
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DOI：
10.1074/jbc.m706741200
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发表时间：
2008-02-22
影响因子：
4.8
通讯作者：
Jung, Heinrich
中科院分区：
文献类型：
--
作者：
Hilger, Daniel;Bohm, Maret;Jung, Heinrich
Na+/溶质同向转运体家族包括400多个原核和真核来源的成员。以大肠杆菌的Na+/脯氨酸转运蛋白PutP为模型，研究了两个保守残基Ser-340和Thr-341的作用，以深入了解该家族成员催化的转运机制。这些氨基酸的取代显著改变了含有PutP变体的细胞和蛋白脂质体的转运动力学。特别是，Na+和Li+的表观亲和力降低了2个数量级或更多。脯氨酸结合也受到影响，尽管程度低于离子结合。因此，在位置341处存在羟基对于高亲和力配体结合是必需的。此外，位于位置340或341的Cys与不同极性的巯基试剂反应，表明可从水相接近。此外，Cys交联表明残基与先前显示对配体结合至关重要的其他氨基酸的接近。由于这些原因，建议Ser-340和Thr-341位于配体易位途径中。此外，有人提出，Thr-341的侧链直接参与Na+结合。
The Na+/solute symporter family comprises more than 400 members of pro- and eukaryotic origin. Using the Na+/proline transporter PutP of Escherichia coli as a model, the role of two conserved residues, Ser-340 and Thr-341, is investigated to obtain insights into the mechanism of transport catalyzed by members of this family. Substitution of these amino acids alters the transport kinetics of cells and proteoliposomes containing the PutP variants significantly. In particular, the apparent affinities for Na+ and Li+ are reduced by 2 orders of magnitude or more. Also proline binding is affected, albeit to a lesser extent than ion binding. Thereby, the presence of a hydroxyl group at position 341 is essential for high affinity ligand binding. Furthermore, Cys placed at position 340 or 341 reacts with sulfhydryl reagents of different polarity, indicating accessibility from the water phase. In addition, Cys cross-linking suggests proximity of the residues to other amino acids previously shown to be crucial for ligand binding. For these reasons it is suggested that Ser-340 and Thr-341 are located in a ligand translocation pathway. Furthermore, it is proposed that the side chain of Thr-341 directly participates in Na+ binding.