Studies on the Molecular Structure and Function of Tetracycline Efflux Protein

自1990年以来，在细菌中发现了许多药物外排蛋白。因此，四环素外排蛋白（tete）作为研究药物外排蛋白分子机制的独特蛋白，其分子生物学研究显得尤为重要。本研究主要通过基因工程方法揭示Tet的分子机制。在哺乳动物葡萄糖转运体、细菌糖/H^+同运体和细菌药物输出体等二级膜转运体中，有一个众所周知的序列基序广泛保守，被描述为GXXSDRXGRR。然而，这个基序在转运体功能中的确切作用仍然未知。我们首先利用定点诱变技术研究了该基序的作用。因此，第5个Asp66和第9个Arg70对该功能至关重要。对于Arg70， Lys70突变体保留了约30%的野生型转运活性，而除Cys70突变体外，其余中性或酸性残基突变体均丧失了转运活性。Cys70突变体保持了与Lys70突变体相当的活性。Cys70突变体出人意料的高活性是由于巯基与一个二价阳离子形成，充当阳离子侧链。在该基序的8个突变体中，只有Cys65和Cys70突变体被NEM灭活。添加四环素刺激了NEM与这些突变体的结合，表明位于细胞质表面的基元通过底物暴露在培养基中。相比之下，NEM与位于周质表面的Cys97的结合被四环素抑制，表明周质残基被底物诱导的构象变化所隐藏。这些观察结果证实了我们的四环素/H^+反端口模型，其中四环素使Tet蛋白形成内向开放/外向封闭的构象。

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.