Substrate recognition and proton coupling by a bacterial member of solute carrier family 17.

The solute carrier 17 family transports diverse organic anions using two distinct modes of coupling to a source of energy. Transporters that package glutamate and nucleotide into secretory vesicles for regulated release by exocytosis are driven by membrane potential but subject to allosteric regulation by H+ and Cl−. Other solute carrier 17 members including the lysosomal sialic acid exporter couple the flux of organic anion to cotransport of H+. To begin to understand how similar proteins can perform such different functions, we have studied Escherichia coli DgoT, a H+/galactonate cotransporter. A recent structure of DgoT showed many residues contacting D-galactonate, and we now find that they do not tolerate even conservative substitutions. In contrast, the closely related lysosomal H+/sialic acid cotransporter Sialin tolerates similar mutations, consistent with its recognition of diverse substrates with relatively low affinity. We also find that despite coupling to H+, DgoT transports more rapidly but with lower apparent affinity at high pH. Indeed, membrane potential can drive uptake, indicating electrogenic transport and suggesting a H+:galactonate stoichiometry >1. Located in a polar pocket of the N-terminal helical bundle, Asp46 and Glu133 are each required for net flux by DgoT, but the E133Q mutant exhibits robust exchange activity and rescues exchange by D46N, suggesting that these two residues operate in series to translocate protons. E133Q also shifts the pH sensitivity of exchange by DgoT, supporting a central role for the highly conserved TM4 glutamate in H+ coupling by DgoT.

溶质载体17家族利用两种不同的与能量源耦合的模式运输多种有机阴离子。将谷氨酸和核苷酸包装到分泌囊泡中以便通过胞吐作用进行调节性释放的转运蛋白是由膜电位驱动的，但受到H⁺和Cl⁻的变构调节。其他溶质载体17成员，包括溶酶体唾液酸输出蛋白，将有机阴离子的通量与H⁺的协同转运相耦合。为了开始了解相似的蛋白质如何能够执行如此不同的功能，我们研究了大肠杆菌的DgoT，一种H⁺/半乳糖酸协同转运蛋白。DgoT的一个近期结构显示许多残基与D - 半乳糖酸接触，我们现在发现它们甚至不能耐受保守性替换。相比之下，密切相关的溶酶体H⁺/唾液酸协同转运蛋白Sialin能耐受类似的突变，这与其以相对较低的亲和力识别多种底物是一致的。我们还发现，尽管与H⁺耦合，DgoT在高pH值下运输速度更快，但表观亲和力较低。实际上，膜电位能够驱动摄取，表明是生电转运，并提示H⁺:半乳糖酸的化学计量比>1。天冬氨酸46和谷氨酸133位于N端螺旋束的一个极性口袋中，它们各自都是DgoT净通量所必需的，但E133Q突变体表现出强大的交换活性并挽救D46N的交换，这表明这两个残基串联作用以转运质子。E133Q还改变了DgoT交换的pH敏感性，支持了高度保守的跨膜区4的谷氨酸在DgoT的H⁺耦合中的核心作用。