Department of Biosystems Science and Engineering, Eidgenössische Technische Hochschule Zurich, 4058 Basel, Switzerland.
Proc Natl Acad Sci U S A. 2011 Oct 25;108(43):E890-8. doi: 10.1073/pnas.1109597108. Epub 2011 Oct 10.
BetP, a trimeric Na(+)-coupled betaine symporter, senses hyperosmotic stress via its cytoplasmic C-terminal domain and regulates transport activity in dependence of the cytoplasmic K(+)-concentration. This transport regulation of BetP depends on a sophisticated interaction network. Using single-molecule force spectroscopy we structurally localize and quantify these interactions changing on K(+)-dependent transport activation and substrate-binding. K(+) significantly strengthened all interactions, modulated lifetimes of functionally important structural regions, and increased the mechanical rigidity of the symporter. Substrate-binding could modulate, but not establish most of these K(+)-dependent interactions. A pronounced effect triggered by K(+) was observed at the periplasmic helical loop EH2. Tryptophan quenching experiments revealed that elevated K(+)-concentrations akin to those BetP encounters during hyperosmotic stress trigger the formation of a periplasmic second betaine-binding (S2) site, which was found to be at a similar position reported previously for the BetP homologue CaiT. In BetP, the presence of the S2 site strengthened the interaction between EH2, transmembrane α-helix 12 and the K(+)-sensing C-terminal domain resulting in a K(+)-dependent cooperative betaine-binding.
BetP 是一种三聚体 Na(+)-偶联的甜菜碱转运体，通过其细胞质 C 末端结构域感知高渗胁迫，并依赖细胞质 K(+)浓度调节转运活性。这种 BetP 的转运调节依赖于一个复杂的相互作用网络。我们使用单分子力谱技术对这些在 K(+)依赖的转运激活和底物结合过程中发生变化的相互作用进行结构定位和定量。K(+)显著增强了所有相互作用，调节了功能重要的结构区域的寿命，并增加了转运体的机械刚性。底物结合可以调节，但不能建立大多数这些依赖于 K(+)的相互作用。在周质螺旋环 EH2 处观察到 K(+)触发的明显效应。色氨酸猝灭实验表明，类似于 BetP 在高渗胁迫下遇到的高 K(+)浓度会触发形成第二个周质甜菜碱结合 (S2) 位点，该位点的位置与之前报道的 CaiT 同源物的位置相似。在 BetP 中，S2 位点的存在增强了 EH2、跨膜 α-螺旋 12 和 K(+)感应 C 末端结构域之间的相互作用，导致 K(+)依赖性协同甜菜碱结合。
