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药物外排蛋白。因此，四环素外排蛋白（泰特）作为一类药物外排蛋白的研究范式，其分子生物学研究显得尤为重要。泰特在哺乳动物葡萄糖转运蛋白、细菌糖/H^+共转运蛋白和细菌药物输出蛋白等次级膜转运蛋白中有一个保守的序列基序，命名为GXXSDRXGRR，但该基序在转运蛋白功能中的确切作用尚不清楚。本研究首先利用定点突变技术研究了该基序的作用。因此，第5个Asp 66和第9个Arg 70对该功能至关重要。对于Arg 70，Lys 70突变体保留了约30%的野生型转运活性，而除了Cys 70突变体之外，中性或酸性残基的突变体丧失了转运活性。Cys 70突变体保留了与Lys 70突变体相当的活性。Cys 70突变体的活性出乎意料的高是由于与二价阳离子形成硫醇盐作为阳离子侧链，在该基序的8个Cys突变体中，只有Cys 65和Cys 70突变体被NEM失活，加入四环素刺激NEM与这些突变体的结合，表明位于细胞质表面的基序通过底物暴露于培养基。与此相反，NEM结合到Cys 97，这是位于周质表面上，被抑制四环素，表明周质残留物是隐藏的底物诱导的构象变化。这些观察结果证实了我们的四环素/H^+反向转运模型，即四环素使泰特蛋白质形成内部开放/外部封闭的构象。

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.