Studies on the Molecular Structure and Function of Tetracycline Efflux Protein

自1990年以来，在细菌中发现了大量的药物外排蛋白。因此，四环素外排蛋白(Tet)是目前研究其分子机制的唯一蛋白质，作为这类药物外排蛋白的范例，其分子生物学研究显得尤为重要。在本研究中，我们主要通过基因工程的方法揭示了Tet的分子机制。在哺乳动物葡萄糖转运蛋白、细菌糖/H~+转运蛋白和细菌药物出口蛋白等次生膜转运蛋白中存在一个广为人知的序列基序，被描述为GXXSDRXGRR，但该基序在转运蛋白功能中的确切作用尚不清楚。我们首先利用定点突变技术研究了该基序在本研究中的作用。因此，第5个Asp66和第9个Arg70对于该功能是必不可少的。对于Arg70，Lys70突变体保留了约30%的野生型转运活性，而对中性或酸性残基的突变体失去了转运活性，但Cys70突变体除外。Cys70突变体保持了与Lys70突变体相当的活性。在8个Cys70突变体中，只有Cys65和Cys70突变体被NEM失活。四环素的加入刺激了NEM与这些突变体的结合，表明该基序位于细胞质表面的基序被底物暴露在介质中。相反，四环素抑制了NEM与位于周质表面的Cys97的结合，这表明底物诱导的构象变化掩盖了周质残基。这些观察结果证实了我们的四环素/H^+逆向转运模型，在该模型中，四环素使Tet蛋白成为内开放/外封闭的构象。

Since 1990, there have been a lot of drug efflux proteins found in bacteria. So, the molecular biological studies on tetracycline efflux protein (Tet) , which is now a unique protein of which molecular mechanisms are studied, becomes more important as a paradigm of such drug efflux proteins. In this study, we revealed the molecular mechanism of Tet mainly by gene engineering methods.There is a well-known sequence motif widely conserved in secondary membrane transporters such as mammalian glucose transporters, bacterial sugar/H^+ symporters and bacterial drug exporters, which is depicted as GXXSDRXGRR.However, the precise role of this motif in the transporter function is still unknown. We first studied the role of this motif in this study by using site-directed mutagenesis technique. As a result, the 5th Asp66 and 9th Arg70 are essential for the function. As to Arg70, Lys70 mutant retained about 30% the wild type transport activity, whereas the mutants to a neutral or acidic residues lost the transport activity except for Cys70 mutant. Cys70 mutant retained the activity comparable to Lys70 mutant. The unexpectedly high activity of Cys70 mutant is due to the mercaptide formation with a divalent cation which acts as a cationic side chain.Among 8 Cys mutants of this motif, only Cys65 and Cys70 mutants were inactivated by NEM.The binding of NEM to these mutants was stimulated by addition of tetracycline, indicating that the motif, which is located on the cytoplasmic surface, is exposed to the medium by the substrate. In contrast, NEM binding to Cys97, which is located on the periplasmic surface, was inhibited by tetracycline, indicating that the periplasmic residue is hidden by a substrate-induced conformational change. These observations confirm our model for tetracycline/H^+ antiport, in which tetracycline makes Tet protein an inside-open/outside-closed conformation.