Asymmetric protonation of EmrE.
Asymmetric protonation of EmrE.
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DOI：
10.1085/jgp.201511404
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发表时间：
2015-12
期刊：
影响因子：
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通讯作者：
Henzler-Wildman KA
中科院分区：
文献类型：
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作者：
Morrison EA;Robinson AE;Liu Y;Henzler-Wildman KA
活性位点谷氨酸的14个残基的同型二聚体多药物转运蛋白EmrE具有不同的宏观pKa值，提出了新的问题，关于耦合质子输入药物流出。小型多药耐药转运蛋白EmrE是一种同源二聚体，它利用质子动力提供的能量驱动药物底物的外排。它的"活性位点"残基谷氨酸14（Glu14）从每个亚基的pKa值必须保持在生理pH值左右，以有效地耦合质子进口到药物出口在体内。为了评估EmrE的质子化，用1H-15N TROSY-HSQC核磁共振（NMR）光谱进行pH滴定。这些光谱分析表明，Glu14残基的不对称pKa值在45 ° C下为7.0 ± 0.1和8.2 ± 0.3，在25 ° C下为6.8 ± 0.1和8.5 ± 0.2。这些pKa值大幅增加与溶剂暴露的谷氨酸盐的典型pKa值相比，但在公布的Glu14 pKa值的范围内，从底物结合和转运试验的pH依赖性推断。活性位点突变体E14 D-EmrE的pKa值低于生理pH范围，与其受损的转运活性一致。的NMR光谱表明，质子化状态的活性位点Glu14残基确定的全球结构和向内和向外面向EmrE之间的构象交换率。因此，不对称活性位点Glu14残基的pKa值是质子输入与多药外排正确偶联的关键。然而，这些结果提出了关于耦合机制的新问题，因为它们表明EmrE存在于中性pH附近的质子化状态的混合物中，并且可以在多个不同的质子化状态中向内和向外的形式之间相互转换。
The active-site glutamate 14 residues of the homodimeric multidrug transporter EmrE have distinct macroscopic pKa values, raising new questions regarding the coupling of proton import to drug efflux. The small multidrug resistance transporter EmrE is a homodimer that uses energy provided by the proton motive force to drive the efflux of drug substrates. The pKa values of its “active-site” residues—glutamate 14 (Glu14) from each subunit—must be poised around physiological pH values to efficiently couple proton import to drug export in vivo. To assess the protonation of EmrE, pH titrations were conducted with 1H-15N TROSY-HSQC nuclear magnetic resonance (NMR) spectra. Analysis of these spectra indicates that the Glu14 residues have asymmetric pKa values of 7.0 ± 0.1 and 8.2 ± 0.3 at 45°C and 6.8 ± 0.1 and 8.5 ± 0.2 at 25°C. These pKa values are substantially increased compared with typical pKa values for solvent-exposed glutamates but are within the range of published Glu14 pKa values inferred from the pH dependence of substrate binding and transport assays. The active-site mutant, E14D-EmrE, has pKa values below the physiological pH range, consistent with its impaired transport activity. The NMR spectra demonstrate that the protonation states of the active-site Glu14 residues determine both the global structure and the rate of conformational exchange between inward- and outward-facing EmrE. Thus, the pKa values of the asymmetric active-site Glu14 residues are key for proper coupling of proton import to multidrug efflux. However, the results raise new questions regarding the coupling mechanism because they show that EmrE exists in a mixture of protonation states near neutral pH and can interconvert between inward- and outward-facing forms in multiple different protonation states.