Asymmetric protonation of glutamate residues drives a preferred transport pathway in EmrE
Asymmetric protonation of glutamate residues drives a preferred transport pathway in EmrE
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DOI：
10.1073/pnas.2110790118
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发表时间：
2021-10-12
影响因子：
11.1
通讯作者：
Traaseth, Nathaniel J.
中科院分区：
文献类型：
--
作者：
Li, Jianping;Her, Ampon Sae;Traaseth, Nathaniel J.
EmrE是一种大肠杆菌多药外排泵，是小多药耐药（SMR）家族的成员，通过利用质子动力的能量将药物作为同源二聚体转运。SMR家族转运蛋白在跨膜1（EmrE中的Glu14）中含有保守的谷氨酸残基，其是结合质子和药物所需的。然而，二聚体中两个谷氨酸残基的质子偶联转运机制仍然没有得到解决。在这里，我们使用NMR光谱来确定酸解离常数（pKa）的野生型EmrE和异源二聚体含有一个或两个Glu14残基的二聚体。对于野生型EmrE，我们测量了化学位移的羧基侧链的Glu14使用固态NMR在脂质双层，并获得明确的证据上存在的不对称质子化状态。随后测量的pKa值的异源二聚体与一个单一的Glu14残基没有显着差异，异源二聚体与两个Glu14残基，支持一个模型，其中两个Glu14残基具有独立的pKa值，并没有静电耦合。这些见解支持在二聚体的每个单体中具有明确定义的质子化状态的转运途径，包括优选的面向细胞质的状态，其中在E细胞质中的pH条件下，Glu 14在单体A中去质子化，在单体B中质子化。杆菌我们的研究结果还导致了一个模型，无跳交换，提出了如何交换器与构象依赖的pKa值减少质子泄漏。该模型与SMR家族和由反向重复结构域组成的转运蛋白相关。
EmrE is an Escherichia coli multidrug efflux pump and member of the small multidrug resistance (SMR) family that transports drugs as a homodimer by harnessing energy from the proton motive force. SMR family transporters contain a conserved glutamate residue in transmembrane 1 (Glu14 in EmrE) that is required for binding protons and drugs. Yet the mechanism underlying proton-coupled transport by the two glutamate residues in the dimer remains unresolved. Here, we used NMR spectroscopy to determine acid dissociation constants (pKa) for wild-type EmrE and heterodimers containing one or two Glu14 residues in the dimer. For wild-type EmrE, we measured chemical shifts of the carboxyl side chain of Glu14 using solid-state NMR in lipid bilayers and obtained unambiguous evidence on the existence of asymmetric protonation states. Subsequent measurements of pKa values for heterodimers with a single Glu14 residue showed no significant differences from heterodimers with two Glu14 residues, supporting a model where the two Glu14 residues have independent pKa values and are not electrostatically coupled. These insights support a transport pathway with well-defined protonation states in each monomer of the dimer, including a preferred cytoplasmic-facing state where Glu14 is deprotonated in monomer A and protonated in monomer B under pH conditions in the cytoplasm of E. coli. Our findings also lead to a model, hop-free exchange, which proposes how exchangers with conformation-dependent pKa values reduce proton leakage. This model is relevant to the SMR family and transporters comprised of inverted repeat domains.