Intrinsic Conformational Plasticity of Native EmrE Provides a Pathway for Multidrug Resistance
Intrinsic Conformational Plasticity of Native EmrE Provides a Pathway for Multidrug Resistance
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DOI：
10.1021/ja503145x
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发表时间：
2014-06-04
影响因子：
15
通讯作者：
Traaseth, Nathaniel J.
中科院分区：
文献类型：
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作者：
Cho, Min-Kyu;Gayen, Anindita;Traaseth, Nathaniel J.
EmrE是一种多药耐药外排泵，对多种抗生素和防腐剂具有特异性。为了获得对编码广泛特异性的天然状态的属性的原子尺度洞察，我们在脂质双层和双胞中使用了溶液和固态NMR方法的混合。我们的研究结果表明，天然EmrE二聚体在37 ℃下以类似于300 s（-1）的交换速率（k（ex））在向内和向外的结构构象之间振荡（毫秒运动），这是相对于四苯基磷（TPP+）底物结合形式的蛋白质快50倍。这些可观察到的定量证据表明，TPP+运输循环中的限速步骤不是在不存在药物的情况下的外向-内向构象变化。此外，使用差示扫描量热法，我们发现凝胶到液晶相转变的宽度在TPP+底物不存在下比其存在下宽2摄氏度，这表明转运动力学的变化可以影响膜的相性质。有趣的是，用交联的EmrE进行的实验表明，毫秒级的向内开放到向外开放的动力学并不是加宽的罪魁祸首。相反，量热法和NMR数据支持的结论是，更快的时间尺度的结构动力学（纳秒-微秒）的来源，因此赋予构象可塑性的天然EmrE能够结合结构不同的基板。这些研究结果提供了一个明确的例子，如何在膜蛋白转运蛋白的结构动力学之间的药物自由和绑定状态的差异可以有一个直接的影响，在一个变构的方式对脂质双层的物理性质。
EmrE is a multidrug resistance efflux pump with specificity to a wide range of antibiotics and antiseptics. To obtain atomic-scale insight into the attributes of the native state that encodes the broad specificity, we used a hybrid of solution and solid-state NMR methods in lipid bilayers and bicelles. Our results indicate that the native EmrE dimer oscillates between inward and outward facing structural conformations at an exchange rate (k(ex)) of similar to 300 s(-1) at 37 degrees C (millisecond motions), which is 50-fold faster relative to the tetraphenylphosphonium (TPP+) substrate-bound form of the protein. These observables provide quantitative evidence that the rate-limiting step in the TPP+ transport cycle is not the outward-inward conformational change in the absence of drug. In addition, using differential scanning calorimetry, we found that the width of the gel-to-liquid crystalline phase transition was 2 degrees C broader in the absence of the TPP+ substrate versus its presence, which suggested that changes in transporter dynamics can impact the phase properties of the membrane. Interestingly, experiments with cross-linked EmrE showed that the millisecond inward-open to outward-open dynamics was not the culprit of the broadening. Instead, the calorimetry and NMR data supported the conclusion that faster time scale structural dynamics (nanosecond-microsecond) were the source and therefore impart the conformationally plastic character of native EmrE capable of binding structurally diverse substrates. These findings provide a clear example how differences in membrane protein transporter structural dynamics between drug-free and bound states can have a direct impact on the physical properties of the lipid bilayer in an allosteric fashion.