Bacterial membrane porins facilitate the translocation of small molecules while restricting large molecules, and this mechanism remains elusive at the molecular level. Here, we investigate the selective uptake of large cyclic sugars across an unusual passive membrane transporter, CymA, comprising a charged zone and a constricting N terminus segment. Using a combination of electrical recordings, protein mutagenesis and molecular dynamics simulations, we establish substrate translocation across CymA governed by the electrostatic pore properties and conformational dynamics of the constriction segment. Notably, we show that the variation in pH of the environment resulted in reversible modulation of the substrate binding site in the pore, thereby regulating charge-selective transport of cationic, anionic and neutral cyclic sugars. The quantitative kinetics of cyclic sugar translocation across CymA obtained in electrical recordings at different pHs are comparable with molecular dynamics simulations that revealed the transport pathway, energetics and favorable affinity sites in the pore for substrate binding. We further define the molecular basis of cyclic sugar translocation and establish that the constriction segment is flexible and can reside inside or outside the pore, regulating substrate translocation distinct from the ligand-gated transport mechanism. Our study provides novel insights into energy-independent large molecular membrane transport for targeted drug design strategies.

中文翻译：

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构象灵活性驱动电荷选择性底物跨细菌转运蛋白易位


细菌膜孔蛋白促进小分子的易位，同时限制大分子，这种机制在分子水平上仍然难以捉摸。在这里，我们研究了大环糖通过一种不寻常的被动膜转运蛋白 CymA 的选择性摄取，该转运蛋白包含带电区和收缩的 N 末端片段。结合电记录、蛋白质诱变和分子动力学模拟，我们建立了由静电孔特性和收缩片段构象动力学控制的跨 CymA 的底物易位。值得注意的是，我们发现环境 pH 值的变化导致孔中底物结合位点的可逆调节，从而调节阳离子、阴离子和中性环糖的电荷选择性运输。在不同 pH 值下的电记录中获得的环糖易位跨 CymA 的定量动力学与分子动力学模拟相当，分子动力学模拟揭示了孔中底物结合的运输途径、能量学和有利的亲和位点。我们进一步定义了环状糖易位的分子基础，并确定收缩片段是灵活的，可以驻留在孔内或孔外，调节底物易位，与配体门控运输机制不同。我们的研究为靶向药物设计策略的能量独立大分子膜运输提供了新的见解。