On the mechanism of substrate specificity by resistance nodulation division (RND)-type multidrug resistance pumps:: the large periplasmic loops of MexD from Pseudomonas aeruginosa are involved in substrate recognition
On the mechanism of substrate specificity by resistance nodulation division (RND)-type multidrug resistance pumps:: the large periplasmic loops of MexD from Pseudomonas aeruginosa are involved in substrate recognition
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DOI：
10.1046/j.1365-2958.2002.03223.x
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发表时间：
2002-11-01
影响因子：
3.6
通讯作者：
Lomovskaya, O
中科院分区：
文献类型：
--
作者：
Mao, WM;Warren, MS;Lomovskaya, O
革兰氏阴性菌的三方外排系统含有一个属于耐药结瘤分裂（RND）超家族的内膜转运蛋白，可以挤出多种结构多样的化合物。为了深入了解这些多药耐药（MDR）转运体识别底物的分子机制，我们从铜绿假单胞菌中分离出改变mexd - oprj泵底物特异性的自发突变。这些突变使泵能够挤出通常不运输的-内酰胺类抗生素卡比西林。所有的氨基酸替换都被映射到RND的大质周环（LPLs）上，MexD。Q34K、E89K、A292V和P328L在第一个LPL中发现，位于跨膜结构域（TMD） 1和2之间，而F608S和N673K在第二个LPL中发现，位于TMD7和TMD8之间。这些突变对mexd - oprj介导的许多其他底物的转运也有重大影响。随后用半胱氨酸替代上述鉴定的氨基酸残基，使mexd - oprj容易受到硫醇反应剂MIANS的抑制。有趣的是，MIANS抑制泵的某些底物（pyronin, EtBr）的运输，但不抑制其他底物（ANS, Leu-Nap）的运输。我们的结果表明，MexD的质周环的精确结构决定了单个底物的运输速率。这些结果与假设一致，即在RND转运体的情况下，LPLs直接参与底物识别，并且包含多种结构不同的化合物的多个相互作用位点。
Tripartite efflux systems of Gram-negative bacteria that contain an inner membrane transporter belonging to the resistance nodulation division (RND) superfamily can extrude a large variety of structurally diverse compounds. To gain an insight into the molecular mechanisms of substrate recognition by these multidrug resistance (MDR) transporters, we isolated spontaneous mutations that altered the substrate specificity of the MexCD-OprJ pump from Pseudomonas aeruginosa. These mutations enabled the pump to extrude the normally non-transported beta-lactam antibiotic carbenicillin. All amino acid substitutions were mapped to the large periplasmic loops (LPLs) of the RND proper, MexD. Q34K, E89K, A292V and P328L were found in the first LPL, located between transmembrane domains (TMD) 1 and 2, whereas F608S and N673K were contained in the second LPL, located between TMD7 and TMD8. These mutations also had a substantial impact on the MexCD-OprJ-mediated transport of numerous other substrates. Subsequent replacement of amino acid residues identified above by cysteines rendered MexCD-OprJ susceptible to inhibition by a thiol-reactive agent, MIANS. Interestingly, MIANS inhibited the transport of some (pyronin, EtBr) but not other (ANS, Leu-Nap) substrates of the pump. Our results suggest that the precise structure of the periplasmic loops of MexD determines the rate of transport of individual substrates. These results are consistent with the hypothesis that, in the case of RND transporters, the LPLs are directly implicated in substrate recognition and contain multiple sites of interaction for various structurally diverse compounds.