Yang Janet G, Chen Hulda Yuchun, Guardado John H, Gardner Maile, Foronda Matthew S
Department of Chemistry, University of San Francisco, San Francisco, California 94117.
bioRxiv. 2025 Jan 20:2025.01.20.633972. doi: 10.1101/2025.01.20.633972.
The MetNI-Q importer, an ATP-binding cassette (ABC) transporter, mediates the uptake of both L- and D- enantiomers of methionine. Original uptake studies show a strong preference for L-Met over D-Met, but the molecular basis of this selectivity is unclear. In this work, we systematically examine substrate discrimination by the MetNI transporter and MetQ substrate binding protein using an array of biophysical and biochemical techniques. Based on the kinetic and thermodynamic parameters of individual intermediates in the transport cycle, we uncover multiple steps in the transport cycle that confer substrate specificity. As in many other ABC importer systems, selectivity is applied at the level of binding to the substrate binding protein: MetQ dictates a 1,000-fold preference for L-Met over D-Met. However, beyond this initial level of selectivity, MetQ displays distinct binding preferences for the MetNI transporter depending on the substrate. We propose that the differences in binding affinities reflect the more favored release of L-Met into the permeation pathway when compared to D-Met. In support of this model, under saturating conditions, MetNI transports L-Met across the lipid bilayer at a faster rate than D-Met. Interestingly, the ATPase activity of the MetNI-Q complex is not modulated by the presence of substrate. Our studies reveal that the MetNI-Q system incorporates two separate steps in tuning methionine uptake to substrate chirality and availability. This method of discrimination ensures the import of the most biologically preferred substrate while also allowing for adaptability to more limiting nutrient conditions.
MetNI-Q 转运体是一种 ATP 结合盒（ABC）转运蛋白，可介导 L-和 D- 蛋氨酸对映体的摄取。最初的摄取研究表明，与 D-蛋氨酸相比，该转运体对 L-蛋氨酸有强烈偏好，但这种选择性的分子基础尚不清楚。在这项研究中，我们使用一系列生物物理和生化技术，系统地研究了 MetNI 转运体和 MetQ 底物结合蛋白对底物的识别情况。基于转运循环中各个中间体的动力学和热力学参数，我们发现了转运循环中赋予底物特异性的多个步骤。与许多其他 ABC 导入系统一样，选择性在与底物结合蛋白结合的水平上起作用：MetQ 对 L-蛋氨酸的偏好比对 D-蛋氨酸高 1000 倍。然而，除了这种初始的选择性水平外，MetQ 对 MetNI 转运体的结合偏好因底物而异。我们提出，结合亲和力的差异反映了与 D-蛋氨酸相比，L-蛋氨酸更有利于释放到渗透途径中。支持这一模型的是，在饱和条件下，MetNI 跨脂质双层转运 L-蛋氨酸的速度比 D-蛋氨酸快。有趣的是，MetNI-Q 复合物的 ATP 酶活性不受底物存在的调节。我们的研究表明，MetNI-Q 系统在调节蛋氨酸摄取以适应底物手性和可用性方面包含两个独立的步骤。这种区分方法可确保导入最符合生物学需求的底物，同时也允许适应更有限的营养条件。
