Bippes Christian A, Zeltina Antra, Casagrande Fabio, Ratera Merce, Palacin Manuel, Muller Daniel J, Fotiadis Dimitrios
Biotechnology Center, Technische Universität Dresden, D-01307 Dresden, Germany.
J Biol Chem. 2009 Jul 10;284(28):18651-63. doi: 10.1074/jbc.M109.004267. Epub 2009 May 6.
We used single molecule dynamic force spectroscopy to unfold individual serine/threonine antiporters SteT from Bacillus subtilis. The unfolding force patterns revealed interactions and energy barriers that stabilized structural segments of SteT. Substrate binding did not establish strong localized interactions but appeared to be facilitated by the formation of weak interactions with several structural segments. Upon substrate binding, all energy barriers of the antiporter changed thereby describing the transition from brittle mechanical properties of SteT in the unbound state to structurally flexible conformations in the substrate-bound state. The lifetime of the unbound state was much shorter than that of the substrate-bound state. This leads to the conclusion that the unbound state of SteT shows a reduced conformational flexibility to facilitate specific substrate binding and a reduced kinetic stability to enable rapid switching to the bound state. In contrast, the bound state of SteT showed an increased conformational flexibility and kinetic stability such as required to enable transport of substrate across the cell membrane. This result supports the working model of antiporters in which alternate substrate access from one to the other membrane surface occurs in the substrate-bound state.
我们使用单分子动态力谱技术来展开来自枯草芽孢杆菌的单个丝氨酸/苏氨酸反向转运蛋白SteT。展开力模式揭示了稳定SteT结构片段的相互作用和能量屏障。底物结合并未建立强烈的局部相互作用，但似乎通过与几个结构片段形成弱相互作用而得到促进。底物结合后，反向转运蛋白的所有能量屏障都发生了变化，从而描述了从SteT在未结合状态下的脆性机械性能到底物结合状态下结构灵活构象的转变。未结合状态的寿命比底物结合状态的寿命短得多。这得出的结论是，SteT的未结合状态显示出降低的构象灵活性以促进特定底物结合，以及降低的动力学稳定性以实现快速切换到结合状态。相比之下，SteT的结合状态显示出增加的构象灵活性和动力学稳定性，这是使底物跨细胞膜运输所必需的。这一结果支持了反向转运蛋白的工作模型，即在底物结合状态下，底物从一个膜表面交替进入另一个膜表面。
