Elevator mechanism dynamics in a sodium-coupled dicarboxylate transporter
Elevator mechanism dynamics in a sodium-coupled dicarboxylate transporter
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钠偶联二羧酸转运蛋白中的电梯机制动力学
DOI：
10.1101/2022.05.01.490196
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发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Kinz-Thompson C
中科院分区：
文献类型：
--
作者：
Kinz-Thompson C
VcINDY是来自霍乱弧菌的钠依赖性二羧酸转运蛋白，负责C4-和C5-羧酸摄入细胞。VcINDY如何以物理方式使底物跨膜移动，并以能量有效的方式移动的分子机制尚不清楚。在这里，我们使用单分子荧光共振能量转移实验直接观察VcINDY在脂质双层中转运底物所需的各个机械步骤，然后测试运输循环机械模型的关键预测。我们的数据提供了第一个直接的证据表明，VcINDY经历随机，电梯型构象运动，使基板易位。动力学分析表明，VcINDY同二聚体的两个原聚体以非合作的方式进行这些运动，从而催化两个独立的运输反应。这些运动的相对底物独立性支持VcINDY运输循环使用合作结合机制保持严格的共底物偶联的概念。最后，热力学模型提供了深入了解如何这样一个合作的结合机制提供了一个通用的方法来优化许多二级活性transporters.Significance StatementTransporter蛋白质的运输使用能量移动分子材料进出细胞。为了提高效率，负责移动分子的转运体运动必须经过严格的设计，以避免在不运输任何东西的情况下浪费能量。通过在单分子水平上测量原型转运蛋白（VcINDY）的运动和动力学，这项研究发现了第一个证据，即像VcINDY这样的转运蛋白通过协调不断动态的“电梯型”运动，同时坐在细胞膜上，实现有效的运输。这些令人惊讶的动态转运蛋白的效率，然后通过热力学建模，这解释了如何高度合作，底物结合反应可能已经演变为最大限度地提高转运蛋白效率的最佳策略背后的分子基础。
VcINDY, the sodium-dependent dicarboxylate transporter fromVibrio cholerae, is responsible for C4- and C5-carboxylate uptake into cells. The molecular mechanism of how VcINDY physically moves substrates across the membrane, and does so in an energetically efficient manner, is unclear. Here, we use single-molecule fluorescence resonance energy transfer experiments to directly observe the individual mechanistic steps that VcINDY takes to translocate substrates across a lipid bilayer, and then test key predictions of transport cycle mechanistic models. Our data provide the first direct evidence that VcINDY undergoes stochastic, elevator-type conformational motions that enable substrate translocation. Kinetic analysis suggests that the two protomers of the VcINDY homodimer undergo those motions in a non-cooperative manner, and thus catalyze two independent transport reactions. The relative substrate independence of those motions supports the notion that the VcINDY transport cycle maintains strict co-substrate coupling using a cooperative binding mechanism. Finally, thermodynamic modeling provides insight into how such a cooperative binding mechanism provides a generalized approach to optimizing transport for many secondary active transporters.Significance StatementTransporter proteins use energy to move molecular materials into and out of cells. To be efficient, the transporter motions responsible for moving the molecules must be tightly choreographed to avoid wasting energy without transporting anything. By measuring the motions and kinetics of a prototypical transporter (VcINDY) at the single-molecule level, this study finds the first evidence that transporters like VcINDY achieve efficient transport by coordinating constantly dynamic, “elevator-type” motions while sitting in the cellular membrane. The efficiency of these surprisingly dynamic transporters is then revealed by thermodynamic modeling, which explains the molecular basis behind how highly cooperative, substrate binding reactions may have evolved as the optimal strategy for maximizing transporter efficiency.
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DOI：
10.1111/febs.16158
发表时间：
2022-03
期刊：
The FEBS journal
影响因子：
--
作者：
通讯作者：
--
影响因子：
3.8
作者：
Lolkema, Juke S.;Slotboom, Dirk J.
通讯作者：
Slotboom, Dirk J.
单个大肠杆菌细胞内钠浓度的荧光测量。
影响因子：
3.4
作者：
Chien;M. Leake;R. Berry
通讯作者：
R. Berry
影响因子：
--
作者：
D. Markovich
通讯作者：
D. Markovich
影响因子：
3.4
作者：
van de Meent, Jan-Willem;Bronson, Jonathan E.;Gonzalez, Ruben L., Jr.
通讯作者：
Gonzalez, Ruben L., Jr.