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Intermolecular and Intramolecular Interactions of the Arabidopsis Plasma Membrane Proton Pump Revealed Using a Mass Spectrometry Cleavable Cross-Linker.
Biochemistry ( IF 2.9 ) Pub Date : 2020-05-27 , DOI: 10.1021/acs.biochem.0c00268
Thao T Nguyen 1 , Matthew R Blackburn 1 , Michael R Sussman 1
Affiliation  

In plants and fungi, the plasma membrane proton pump (H+-ATPase) establishes an electrochemical gradient across the plasma membrane, which serves as the driving force for the secondary transport of ions and nutrients across the cell membrane. This is an essential enzyme that functions in many important processes including stomatal movement, cell elongation, and cellular responses to stimuli from hormones, light, and other environmental conditions. Therefore, understanding how the activity of the H+-ATPase is regulated is important to understand how plants adapt to different growth conditions. The autoinhibitory effect of the C-terminal regulatory domain of H+-ATPase is well-established and is thought to be mediated by interactions with the catalytic domains. Here, using the lysine reactive mass spectrometry cleavable cross-linker DSSO, we found that the C-terminal domain of the Arabidopsis H+-ATPase 2 (AHA2) cross-linked extensively with the actuator, nucleotide-binding, and phosphorylation domains, suggesting that the C-terminal domain regulates the catalytic cycle by modulating the relative positions of these domains. Interestingly, several C-terminal cross-links occurred near a predicted proton binding site (Asp-684 in TM6), suggesting that the C-terminal domain may regulate proton efflux. Additionally, cross-links between the C-terminal domain and other domains of AHA2 were detected in a monomeric protein resolved on SDS-PAGE, suggesting that intramolecular interactions may also be involved in the regulation of enzyme activity. Finally, we observed mixed-isotope cross-linking between the C-terminal domain and other domains of 14N-AHA2 (unlabeled) and 15N-AHA2 (labeled), supporting our model that oligomeric H+-ATPase may autoinhibit the neighboring monomer in a “head-to-tail” configuration.

中文翻译:

拟南芥质膜质子泵的分子间和分子内相互作用揭示了使用质谱可裂解交联剂。

在植物和真菌中,质膜质子泵(H + -ATPase)在质膜上建立电化学梯度,这是离子和营养物在细胞膜上二次运输的驱动力。这是一种必不可少的酶,它在许多重要过程中起作用,包括气孔运动,细胞伸长以及细胞对激素,光照和其他环境条件刺激的反应。因此,了解如何调节H + -ATPase的活性对于了解植物如何适应不同的生长条件非常重要。H +的C末端调节域的自抑制作用-ATP酶是公认的,并被认为是通过与催化结构域的相互作用介导的。在这里,使用赖氨酸反应质谱可裂解的交联剂DSSO,我们发现拟南芥H +的C末端结构域-ATPase 2(AHA2)与致动器,核苷酸结合和磷酸化结构域广泛交联,表明C末端结构域通过调节这些结构域的相对位置来调节催化循环。有趣的是,在预计的质子结合位点(TM6中的Asp-684)附近发生了几个C末端交联,这表明C末端结构域可能调节质子流出。另外,在通过SDS-PAGE解析的单体蛋白中检测到了AHA2的C末端结构域与其他结构域之间的交联,这表明分子内相互作用也可能参与了酶活性的调节。最后,我们观察到14 N-AHA2(未标记)和15 N-AHA2的C末端结构域与其他结构域之间的混合同位素交联N-AHA2(标记)支持我们的模型,即低聚H + -ATPase可以“头到尾”构型自动抑制邻近的单体。
更新日期:2020-06-23
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