BglF, the sensor of the bgl system and the β-glucosides permease of Escherichia coli:: Evidence for dimerization and intersubunit phosphotransfer
BglF, the sensor of the bgl system and the β-glucosides permease of Escherichia coli:: Evidence for dimerization and intersubunit phosphotransfer
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DOI：
10.1021/bi9731652
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发表时间：
1998-06-16
期刊：
影响因子：
2.9
通讯作者：
Amster-Choder, O
中科院分区：
文献类型：
--
作者：
Chen, Q;Amster-Choder, O
大肠杆菌BglF蛋白，也称为EIIbgl，是磷酸烯醇化酶依赖性磷酸转移酶系统（PTS）的酶II，其催化β-葡糖苷的转运和磷酸化。此外，BglF具有EII不寻常的能力，通过根据β-葡糖苷的可用性使其磷酸化和去磷酸化来调节转录调节因子BglG的活性。BglF和BglG共同构成了一个新的感觉系统。膜结合传感器BglF具有两个磷酸化位点：位点1接受来自HPr的磷酰基并将其递送至位点2;位点2将磷酰基递送至β-葡糖苷或BglG。在这里，我们提供了几条线的证据BglF的二聚体和生产亚基间磷酸转移内的BglF二聚体的发生。(1)两种失活的BglF突变蛋白，一种缺乏磷酸化位点1，另一种缺乏位点2，彼此互补以允许bglF菌株利用β-葡糖苷。(2)成对的突变体蛋白在体内作为转录抗终止子调节BglG活性中彼此互补。(3)只有当它们存在于相同的膜制备中时，突变蛋白质对才能在体外有效地将磷酰基从HPr转移到β-葡糖苷和BglG。(4)温和提取细胞蛋白，然后进行SDS-PAGE，揭示BglF同源二聚体的存在。BglF的磷酸化形式的一部分也可以作为二聚体从膜中提取。二聚化由膜结合的IICbgl结构域介导，如IICbgl自身和含有该结构域的BglF衍生物的二聚化所示。由于二聚体在还原剂存在下持续存在，它们显然不是通过二硫键结合在一起。相反，BglF二聚化可能涉及跨膜结构域中残基之间的疏水相互作用。此外，我们表明，BglF二聚化是不调制的β-葡糖苷，因此：不是转移的磷酰基基团远离BglG的运输糖的机制的一部分，在添加β-葡糖苷的生长培养基。
The Escherichia coli BglF protein, also designated EIIbgl, is an enzyme II of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) that catalyzes transport and phosphorylation of beta-glucosides. In addition, BglF has the ability, unusual for an EII, to regulate the activity of a transcriptional regulator, BglG, by phosphorylating and dephosphorylating it according to beta-glucoside availability. Together, BglF and BglG constitute a novel sensory system. The membrane-bound sensor, BglF, has two phosphorylation sites: site 1 accepts a phosphoryl group from HPr and delivers it to site 2; site 2 delivers the phosphoryl group either to beta-glucosides or to BglG. Here, we provide several lines of evidence for the dimerization of BglF and for the occurrence of productive intersubunit phosphotransfer within the BglF dimers. (1) Two inactive BglF mutant proteins, one lacking phosphorylation site 1 and the other lacking site 2, complement one another to allow beta-glucoside utilization by bglF strains. (2) The pairs of mutant proteins complement one another in regulating BglG activity as a transcriptional antiterminator in vivo. (3) Only when they are present in the same membrane preparation do the mutant protein pairs efficiently transfer the phosphoryl group from HPr to beta-glucosides and to BglG in vitro. (4) Gentle extraction of cellular proteins followed by SDS-PAGE reveals the existence of BglF homodimers. A portion of the phosphorylated form of BglF can also be extracted from the membrane as a dimer. Dimerization is mediated by the membrane-bound IICbgl domain, as indicated by the dimerization of IICbgl by itself and of BglF derivatives that contain this domain. Since dimers persist in the presence of a reducing agent, they are apparently not held together by disulfide bonds. Rather, BglF dimerization might involve hydrophobic interactions between residues in the membrane-spanning domain. In addition, we show that BglF dimerization is not modulated by beta-glucosides and is therefore: not part of the mechanism that diverts the phosphoryl group away from BglG to the transported sugar upon addition of beta-glucosides to the growth medium.