Light-driven sodium pumps (NaRs) are unique ion-transporting microbial rhodopsins. The major group of NaRs is characterized by an NDQ motif and has two aspartic acid residues in the central region essential for sodium transport. Here we identify a subgroup of the NDQ rhodopsins bearing an additional glutamic acid residue in the close vicinity to the retinal Schiff base. We thoroughly characterize a member of this subgroup, namely the protein ErNaR from Erythrobacter sp. HL-111 and show that the additional glutamic acid results in almost complete loss of pH sensitivity for sodium-pumping activity, which is in contrast to previously studied NaRs. ErNaR is capable of transporting sodium efficiently even at acidic pH levels. X-ray crystallography and single particle cryo-electron microscopy reveal that the additional glutamic acid residue mediates the connection between the other two Schiff base counterions and strongly interacts with the aspartic acid of the characteristic NDQ motif. Hence, it reduces its pKa. Our findings shed light on a subgroup of NaRs and might serve as a basis for their rational optimization for optogenetics.

光驱动钠泵（NaR）是独特的离子传输微生物视紫红质。 NaR 的主要基团以 NDQ 基序为特征，并且在钠转运所必需的中心区域具有两个天冬氨酸残基。在这里，我们鉴定了 NDQ 视紫红质的一个亚组，在视网膜希夫碱附近带有额外的谷氨酸残基。我们彻底表征了该亚组的一个成员，即来自红杆菌属的蛋白质Er NaR。 HL-111表明，额外的谷氨酸导致钠泵活性的 pH 敏感性几乎完全丧失，这与之前研究的 NaR 形成鲜明对比。即使在酸性 pH 水平下， Er NaR 也能够有效运输钠。 X 射线晶体学和单粒子冷冻电子显微镜表明，额外的谷氨酸残基介导其他两个席夫碱抗衡离子之间的连接，并与特征 NDQ 基序的天冬氨酸强烈相互作用。因此，它降低了其pKa。我们的研究结果揭示了 NaR 的一个亚组，并可能作为其光遗传学合理优化的基础。