Transport-defective mutations alter the conformation of the energy-coupling motif of an outer membrane transporter
Transport-defective mutations alter the conformation of the energy-coupling motif of an outer membrane transporter
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DOI：
10.1021/bi015602p
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发表时间：
2001-11-20
期刊：
影响因子：
2.9
通讯作者：
Cafiso, DS
中科院分区：
文献类型：
--
作者：
Coggshall, KA;Cadieux, N;Cafiso, DS
维生素 B-12 的细菌外膜转运蛋白 BtuB 通过与跨周质膜蛋白 TonB 相互作用获取转运能量。这种与 TonB 的相互作用部分是通过 BtuB 序列中称为 Ton 盒的 N 端片段发生的。在本研究中，使用完整外膜制剂的定点自旋标记来研究野生型 BtuB 和两种转运缺陷突变体 L8P 和 V10P 中 Ton 盒的构象。在野生型蛋白质中，Ton 盒被折叠到转运蛋白的桶中。该片段的构象在转运缺陷突变体 L8P 和 V10P 中显着不同，其中 Ton 盒被发现是柔性的，并且与转运桶分离，更多地暴露于周质。在野生型蛋白质中，维生素 B-12 会诱导 Ton 盒脱离，但将其添加到这些转运缺陷突变体中对 Ton 盒的构象几乎没有或没有产生变化。不改变运输的位置处的脯氨酸取代不会改变 Ton 盒的野生型构象；因此，在位置 8 和 10 处替换脯氨酸对 Ton 盒的对接状态的影响似乎是独特的。这些突变体未能执行 B-12 运输循环可能是 Ton 盒构象改变的结果。
The bacterial outer membrane transporter for vitamin B-12, BtuB, derives its energy for transport by interacting with the trans-periplasmic membrane protein TonB. This interaction with TonB occurs in part through an N-terminal segment in the BtuB sequence called the Ton box. In the present study, site-directed spin labeling of intact outer membrane preparations was used to investigate the conformation of the Ton box in wild-type BtuB and in two transport-defective mutants, L8P and V10P. In the wild-type protein, the Ton box is folded into the barrel of the transporter. The conformation of this segment is dramatically different in the transport-defective mutants L8P and V10P, where the Ton box is found to be flexible, and undocked from the transporter barrel with a greater exposure to the periplasm. In the wild-type protein, vitamin B-12 induces an undocking of the Ton box, but its addition to these transport defective mutants produces little or no change in the conformation of the Ton box. Proline substitutions at positions that do not alter transport do not alter the wild-type conformation of the Ton box; thus, the effect of substituting proline at positions 8 and 10 on the docked state of the Ton box appears to be unique. The failure of these mutants to execute the B-12 transport cycle may be a result of the altered conformation of the Ton box.