The Role of Tryptophans 371 and 395 in the Binding of Antibiotics and the Transport of Sugars by the D-Galactose-H+ Symport Protein (GalP) from Escherichia coli(*)
The Role of Tryptophans 371 and 395 in the Binding of Antibiotics and the Transport of Sugars by the D-Galactose-H+ Symport Protein (GalP) from Escherichia coli(*)
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色氨酸 371 和 395 在抗生素结合和大肠杆菌 D-半乳糖-H 同轴转运蛋白 (GalP) 转运糖中的作用(*)
DOI：
10.1074/jbc.270.51.30359
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
P. Henderson
中科院分区：
文献类型：
--
作者：
T. McDonald;A. Walmsley;G. E. Martin;P. Henderson
研究了大肠杆菌D-半乳糖-H+同向转运体（GalP）与抑制性抗生素细胞松弛素B和forskolin以及底物D-半乳糖和H+之间的相互作用，并对突变体Trp-371 →Phe和Trp-395 → Phe进行了结构-活性关系分析。两种突变均不能阻止[4- 3 H]细胞松弛素B或3-[125 I]碘-4-叠氮苯乙基氨基-7-O-琥珀酰去乙酰基毛喉素（[125 I] APS-毛喉素）的光标记。然而，蛋白质荧光的测量结果表明，这两个残基的结构域，其构象被细胞松弛素B或毛喉素的结合扰动。此外，这两种突变导致的GalP蛋白质的面向内的网站的细胞松弛素B，10-和43倍，分别大幅减少的亲和力，但对毛喉素，0.8-和2.6倍减少，分别在该网站的亲和力几乎没有影响。这两种突变改变了假定的外向（T1）和内向（T2）构象之间的平衡，因此内向型更受青睐。它们还稳定了一种不同的构象状态，即“T3-抗生素”，在这种状态下，蛋白质和抗生素之间的初始相互作用被加强。总的来说，这具有补偿对细胞松弛素B的亲和力降低的作用，使得相应的总Kd值是野生型的0.74倍和3.5倍，同时导致突变体对毛喉素的亲和力分别轻微增加1.5倍和3.2倍。Trp-371 →Phe突变导致内向位点对D-半乳糖的亲和力降低15倍，表明该残基形成了糖结合位点的一部分。相反，Trp-395 →Phe突变对D-半乳糖的内向位点的亲和力没有影响。这些效应可能与Trp-371 →Phe突变体中半乳糖-H+同向转运活性的降低有关，尽管它仍然影响与Trp-395 →Phe突变体相同程度的主动转运。然而，有一个10-20倍的增加Km值为通电运输的D-半乳糖的突变体。
The interactions between the D-galactose-H+ symporter (GalP) from Escherichia coli and the inhibitory antibiotics, cytochalasin B and forskolin, and the substrates, D-galactose and H+, have been investigated for the wild-type protein and the mutants Trp-371 →Phe and Trp-395 → Phe, so that the roles of these residues in the structure-activity relationship could be assessed. Neither mutation prevented photolabeling by either [4-3H]cytochalasin B or by 3-[125I]iodo-4-azidophenethylamido-7-O-succinyldesacetylforskolin ([125I]APS-forskolin). However, measurements of protein fluorescence show that both residues are in structural domains, the conformations of which are perturbed by the binding of cytochalasin B or forskolin. Moreover, both mutations cause a substantial decrease in the affinity of the inward-facing site of the GalP protein for cytochalasin B, 10- and 43-fold, respectively, but have little effect upon the affinity of this site for forskolin, 0.8- and 2.6-fold reductions, respectively. Both these mutations change the equilibrium between the putative outward- (T1) and inward-facing (T2) conformations, so that the inward-facing form is more favored. They also stabilize a different conformational state, “T3-antibiotic,” in which the initial interactions between the protein and antibiotics are tightened. Overall, this has the effect of compensating for the reduction in affinity for cytochalasin B, so that the respective overall K>d values are 0.74- and 3.5-fold that of the wild type, while causing a slight increase, 1.5- and 3.2-fold, respectively, in affinity of the mutants for forskolin. The Trp-371 →Phe mutation causes a 15-fold reduction in the affinity of the inward-facing site for D-galactose, suggesting that this residue forms part of the sugar binding site. In contrast, the Trp-395 →Phe mutation has no effect upon the affinity of the inward-facing site for D-galactose. These effects may be related to the reduction in galactose-H+ symport activity only in the Trp-371 →Phe mutant, although it still effects active transport to the same extent as the Trp395 →Phe mutant. However, there is a 10-20-fold increase in the Kmvalues for energized transport of D-galactose for both mutants.
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Forskolin 特异性抑制细菌半乳糖-H 转运蛋白 GalP。
DOI：
--
发表时间：
1994
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Martin,GE;Seamon,KB;Brown,FM;Shanahan,MF;Roberts,PE;Henderson,PJ
通讯作者：
Henderson,PJ
细胞松弛素 B 干扰由内部和外部糖结合引起的人红细胞葡萄糖转运蛋白的构象变化。
影响因子：
2.9
作者：
King,AP;Tai,PK;Carter-Su,C
通讯作者：
Carter-Su,C
与人红细胞糖转运蛋白结合的平衡配体。
DOI：
--
发表时间：
1987
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Helgerson,AL;Carruthers,A
通讯作者：
Carruthers,A
人红细胞糖转运蛋白的 ATP 调节。
DOI：
--
发表时间：
1986
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Carruthers,A
通讯作者：
Carruthers,A
影响因子：
56.9
作者：
MUECKLER, M;CARUSO, C;LODISH, HF
通讯作者：
LODISH, HF