Interdomain loop mutation Asp190Cys of the tetracycline efflux transporter TetA(B) decreases affinity for substrate.
Interdomain loop mutation Asp190Cys of the tetracycline efflux transporter TetA(B) decreases affinity for substrate.
复制标题
四环素外排转运蛋白 TetA(B) 的域间环突变 Asp190Cys 降低了对底物的亲和力。
DOI：
10.1128/aac.00357-07
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发表时间：
2007
影响因子：
4.9
通讯作者：
Levy,StuartB
中科院分区：
文献类型：
--
作者：
Sapunaric,FrédéricM;Levy,StuartB
TetA（B）是来自Tn10的四环素效应蛋白，具有12个跨越大肠杆菌细胞质膜的预测螺旋（10），是主要易化剂超家族的成员（4）。TetA（B）和相关的四环素反向转运蛋白利用细菌跨膜质子动力输出一个金属-四环素复合物以交换一个H（12），从而降低细胞内四环素浓度。在我们最近对连接TetA（B）（7）和TetA（C）（6）的跨膜螺旋6和7的胞质结构域间环的研究中，我们表明该环含有与四环素efffux活性有关的残基。此外，我们确定TetA（B）的残基Asp190、Glu192和Ser201参与泵的底物特异性（7）。在两种兽用沙门氏菌分离株中TetA（A）结构域间环中三个相邻氨基酸的突变显示底物特异性改变，对米诺环素和甘氨酰环素的敏感性降低（11）。在目前的工作中，我们研究了Asp190 Cys突变对四环素efffux的影响的基础上，由TetA（B）在翻转膜囊泡使用[H3]四环素介导。将特异性Asp190Cys突变（7）和野生型TetB（13）的低拷贝质粒（复制起点pSC101）转化至大肠杆菌DH5中;如前所述制备外翻膜囊泡（3）。使用二喹啉甲酸蛋白质测定（Pierce，Rockford，IL）定量蛋白质含量，并将囊泡等分试样在80 ℃下储存在50mMMOPS（吗啉丙磺酸），pH6.6中.通过吖啶橙子荧光法验证囊泡的反向转运活性。如前所述（7），还使用抗Ct抗体通过蛋白质免疫印迹法测定膜中TetA（B）蛋白的量，并且在每种菌株中显示相同的量。使用Graphpad prism 4软件通过拟合Michaelis-Menton方程来确定Km和Vmax值。在位置190处天冬氨酸的半胱氨酸取代显示平均Km值是野生型的3.8倍，但未产生Vmax的任何修饰（表1）。野生型获得的Km和Vmax值与文献中报道的一致（3、8、9、14）。突变体Asp190Cys对四环素的亲和力较低，表明天冬氨酸残基参与了底物相互作用。可能带负电荷的天冬氨酸与带正电荷的二价金属阳离子-四环素复合物（其为底物）相互作用。引起蛋白质Km变化的取代指示参与底物结合的位置已经用-内酰胺酶TEM 1（1，2）显示。虽然Asp190不是四环素efffux的必需残基（10），但我们清楚地表明，将其改为Cys会降低泵对其底物的亲和力（Km较高）。构成胞质结构域间环的约30个氨基酸的序列在十几个相关四环素外输泵中并不保守（5）。直到最近，这个环一直被认为只是一个将蛋白质的两个部分连接在一起的系绳。我们的生化结果现在支持以前的数据在体内，这一环的TetA（B）四环素转运中有一个意想不到的作用。
TetA (B), the tetracycline efffux protein from Tn10, has 12 predicted-helices that span the Escherichia coli cytoplasmic membrane (10) and is a member of the major facilitator superfamily (4). TetA (B) and related tetracycline antiporters use the bacterial transmembrane proton motive force to export one metal-tetracycline complex in exchange for one H (12), thereby reducing the intracellular tetracycline concentration. In our recent studies of the cytoplasmic interdomain loop connecting transmembrane helices 6 and 7 of TetA (B)(7) and TetA (C)(6), we showed that this loop contains residues implicated in tetracycline efffux activity. Furthermore, we established that residues Asp190, Glu192, and Ser201 of TetA (B) are involved in substrate specificity of the pump (7). Mutations of three adjacent amino acids in the interdomain loop of TetA (A) in two veterinary Salmonella isolates showed an altered substrate specificity with reduced susceptibility to minocycline and glycylcyclines (11). In the present work, we investigated the basis for the effect of the Asp190Cys mutation on tetracycline efffux mediated by TetA (B) in everted membrane vesicles using [H3] tetracycline. The low-copy plasmids (origin of replication pSC101) that specified the Asp190Cys mutation (7) and wild-type TetB (13) were transformed into Escherichia coli DH5; everted membrane vesicles were prepared as described previously (3). The protein content was quantified using the bicinchoninic acid protein assay (Pierce, Rockford, IL), and vesicle aliquots were stored in 50 mM MOPS (morpholinepropanesulfonic acid), pH 6.6, at 80 C. The antiport activity of vesicles was verified by an acridine orange ffuorescence method. The amount of TetA (B) protein in the membranes was also determined by Western immunoblotting using anti-Ct antibody as described previously (7) and showed the same amount in each strain. Graphpad prism 4 software was used to determine the Km and Vmax values by fitting to the Michaelis-Menton equation. The cysteine substitution for aspartate at position 190 showed an average Km value 3.8 times that of the wild type but did not produce any modification in Vmax (Table 1). The Km and Vmax values obtained for the wild type are in agreement with those reported in the literature (3, 8, 9, 14). The lower affinity for tetracycline of the mutant Asp190Cys suggests that the aspartate residue is involved in the substrate interaction. Possibly the negatively charged aspartate interacts with the positively charged divalent metal cation-tetracycline complex which is the substrate. That a substitution causing a change in Km of a protein indicates a position involved in substrate binding has been shown with the-lactamase TEM1 (1, 2). Although Asp190 is not an essential residue for tetracycline efffux (10), we show clearly that changing it to Cys lowered the affinity (higher Km) of the pump for its substrate. The sequence of the approximately 30 amino acids comprising the cytoplasmic interdomain loop is not conserved among the dozen or so related tetracycline efffux pumps (5). This loop has been assumed until recently to be simply a tether holding the two halves of the protein together. Our biochemical results now support previous data in vivo that this loop of TetA (B) has an unexpected role in tetracycline transport.