Oversized galactosides as a probe for conformational dynamics in LacY
Oversized galactosides as a probe for conformational dynamics in LacY
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DOI：
10.1073/pnas.1800706115
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发表时间：
2018-04-17
影响因子：
11.1
通讯作者：
Kaback，H. Ronald
中科院分区：
文献类型：
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作者：
Smirnova，Irina;Kasho，Vladimir;Kaback，H. Ronald
用荧光共振能量转移技术（FRET）研究了α-吡喃半乳糖苷同系物与不同大小和形状的荧光配基的结合动力学。观察到与稳定在向外开放构象（kon= 4-20 μM−1·s−1）的LacY的快速结合，表明即使是比乳糖大得多的配体也能畅通无阻地进入结合位点。解离速率常数（koff）增加的糖苷配基的大小，使Kd值也增加，但保持在微摩尔范围内的每个同系物。Phe 27（螺旋I）通过突出到周质腔中而在糖的途径中形成明显的收缩。然而，用体积较大的Trp替换Phe 27不会在途径中产生障碍，即使对于大的配体也是如此，因为结合动力学保持不变。在周质开口部分封闭的LacY/纳米抗体复合物中也观察到结合位点的高度可及性。值得注意的是，E.表达WT LacY的大肠杆菌催化α-或β-吡喃半乳糖苷与过大的糖苷配基如bodipy或Aldol 518的转运，这可能需要在封闭的中间体内的额外空间。结果证实，LacY特异性是严格针对吡喃半乳糖苷环，也清楚地表明，在周质侧的开口是足够宽，以容纳大的半乳糖苷衍生物在这里测试。我们得出结论，从周质侧进入的基板的实际途径比在周质开放的X射线结构中计算的孔径更宽。
Binding kinetics of α-galactopyranoside homologs with fluorescent aglycones of different sizes and shapes were determined with the lactose permease (LacY) ofEscherichia coliby FRET from Trp151 in the binding site of LacY to the fluorophores. Fast binding was observed with LacY stabilized in an outward-open conformation (kon= 4–20 μM−1·s−1), indicating unobstructed access to the binding site even for ligands that are much larger than lactose. Dissociation rate constants (koff) increase with the size of the aglycone so thatKdvalues also increase but remain in the micromolar range for each homolog. Phe27 (helix I) forms an apparent constriction in the pathway for sugar by protruding into the periplasmic cavity. However, replacement of Phe27 with a bulkier Trp does not create an obstacle in the pathway even for large ligands, since binding kinetics remain unchanged. High accessibility of the binding site is also observed in a LacY/nanobody complex with partially blocked periplasmic opening. Remarkably,E. coliexpressing WT LacY catalyzes transport of α- or β-galactopyranosides with oversized aglycones such as bodipy or Aldol518, which may require an extra space within the occluded intermediate. The results confirm that LacY specificity is strictly directed toward the galactopyranoside ring and also clearly indicate that the opening on the periplasmic side is sufficiently wide to accommodate the large galactoside derivatives tested here. We conclude that the actual pathway for the substrate entering from the periplasmic side is wider than the pore diameter calculated in the periplasmic-open X-ray structures.