Department of Physiology, University of California, Los Angeles, CA 90095.
Department of Chemistry, University of British Columbia, Vancouver, BC V6T 1Z1, Canada.
Proc Natl Acad Sci U S A. 2018 Apr 17;115(16):4146-4151. doi: 10.1073/pnas.1800706115. Epub 2018 Mar 30.
Binding kinetics of α-galactopyranoside homologs with fluorescent aglycones of different sizes and shapes were determined with the lactose permease (LacY) of by FRET from Trp151 in the binding site of LacY to the fluorophores. Fast binding was observed with LacY stabilized in an outward-open conformation ( = 4-20 μM·s), indicating unobstructed access to the binding site even for ligands that are much larger than lactose. Dissociation rate constants () increase with the size of the aglycone so that values 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, expressing 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.
用荧光非糖部分与色氨酸 151 在乳糖通透酶（LacY）结合部位的荧光共振能量转移（FRET），测定了不同大小和形状的α-半乳糖苷类似物与荧光非糖部分的结合动力学。在外向开放构象稳定的 LacY 中观察到快速结合（=4-20μM·s），表明即使对于比乳糖大得多的配体，也能无障碍地进入结合部位。离解速率常数（）随非糖部分的大小而增加，因此值也增加，但对于每个类似物仍保持在微摩尔范围内。苯丙氨酸 27（I 螺旋）通过突入周质腔，在糖的途径中形成明显的收缩。然而，用较大的色氨酸取代苯丙氨酸 27 不会在途径中造成障碍，即使对于大的配体也是如此，因为结合动力学保持不变。在部分阻塞周质开口的 LacY/纳米体复合物中也观察到结合部位的高可及性。值得注意的是，表达 WT LacY 的可催化带有过大非糖部分的α-或β-半乳糖苷的转运，例如 bodipy 或 Aldol518，这可能需要在被阻塞的中间体内额外的空间。结果证实，LacY 的特异性严格指向半乳糖吡喃糖苷环，并且还清楚地表明，周质侧的开口足够宽，可以容纳这里测试的大半乳糖苷衍生物。我们得出结论，从周质侧进入的底物的实际途径比周质开放 X 射线结构中计算的孔径宽。
