Gain of Function Mutations in Membrane Region M2C2 of KtrB Open a Gate Controlling K+ Transport by the KtrAB System from Vibrio alginolyticus
Gain of Function Mutations in Membrane Region M2C2 of KtrB Open a Gate Controlling K+ Transport by the KtrAB System from Vibrio alginolyticus
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DOI：
10.1074/jbc.m109.089870
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发表时间：
2010-04-02
影响因子：
4.8
通讯作者：
Bakker, Evert P.
中科院分区：
文献类型：
--
作者：
Haenelt, Inga;Loechte, Sara;Bakker, Evert P.
KtrB 是 Na+ 依赖性细菌 K+ 摄取系统 KtrAB 的 K+ 易位亚基，由四个 M1PM2 结构域组成，其中 M-1 和 M-2 是跨膜螺旋，P 表示从外部介质折叠回细胞膜的 p 环。跨膜延伸 M-2C 具有 40 个残基，非常长。它由三部分组成，疏水螺旋 M-2C1 和 M-2C3，它们通过非螺旋 M-2C2 区域连接，包含保守的甘氨酸、丙氨酸、丝氨酸、苏氨酸和赖氨酸残基。 M-2C2 中的几个点突变导致 KtrB 从溶藻弧菌中摄取 K+ 的功能大幅增强。这种效应完全是由于 K+ 运输的 V-max 增加所致。 KtrB 引起的 Na+ 易位不受影响。 M-2C2 的部分或完全删除也导致通过 KtrB 吸收 K+ 的 Vmax 值增强。然而，一些缺失变体也表现出更高的 K+ 吸收 Km 值，并且至少一种缺失变体 KtrB(Delta 326-328) 也更快地转运 Na+。 KtrA 的存在并没有抑制任何这些效应。对于缺失变体，这是由于 KtrA 与 KtrB 的结合减少所致。 PhoA 研究表明，M-2C2 在膜内形成柔性结构，允许 M-2C3 定向到细胞质或（人工）定向到周质。这些数据被解释为：(i) M-2C2 区域在 KtrB 的细胞质侧形成控制 K+ 易位的灵活门，以及 (ii) M-2C2 是 KtrA 和 KtrB 之间相互作用所必需的。
KtrB, the K+-translocating subunit of the Na+-dependent bacterial K+ uptake system KtrAB, consists of four M1PM2 domains, in which M-1 and M-2 are transmembrane helices and P indicates a p-loop that folds back from the external medium into the cell membrane. The transmembrane stretch M-2C is, with its 40 residues, unusually long. It consists of three parts, the hydrophobic helices M-2C1 and M-2C3, which are connected by a nonhelical M-2C2 region, containing conserved glycine, alanine, serine, threonine, and lysine residues. Several point mutations in M-2C2 led to a huge gain of function of K+ uptake by KtrB from the bacterium Vibrio alginolyticus. This effect was exclusively due to an increase in V-max for K+ transport. Na+ translocation by KtrB was not affected. Partial to complete deletions of M-2C2 also led to enhanced Vmax values for K+ uptake via KtrB. However, several deletion variants also exhibited higher Km values for K+ uptake and at least one deletion variant, KtrB(Delta 326-328), also transported Na+ faster. The presence of KtrA did not suppress any of these effects. For the deletion variants, this was due to a diminished binding of KtrA to KtrB. PhoA studies indicated that M-2C2 forms a flexible structure within the membrane allowing M-2C3 to be directed either to the cytoplasm or (artificially) to the periplasm. These data are interpreted to mean (i) that region M-2C2 forms a flexible gate controlling K+ translocation at the cytoplasmic side of KtrB, and (ii) that M-2C2 is required for the interaction between KtrA and KtrB.