Chemistry and Molecular Biology, Gothenburg University, 405 30 Gothenburg, Sweden.
Pharmaceutical Technology and Biopharmacy, University of Freiburg, 79104 Freiburg im Breisgau, Germany.
Proc Natl Acad Sci U S A. 2017 May 30;114(22):E4360-E4369. doi: 10.1073/pnas.1614066114. Epub 2017 Feb 21.
The EH-domain-containing protein 2 (EHD2) is a dynamin-related ATPase that confines caveolae to the cell surface by restricting the scission and subsequent endocytosis of these membrane pits. For this, EHD2 is thought to first bind to the membrane, then to oligomerize, and finally to detach, in a stringently regulated mechanistic cycle. It is still unclear how ATP is used in this process and whether membrane binding is coupled to conformational changes in the protein. Here, we show that the regulatory N-terminal residues and the EH domain keep the EHD2 dimer in an autoinhibited conformation in solution. By significantly advancing the use of infrared reflection-absorption spectroscopy, we demonstrate that EHD2 adopts an open conformation by tilting the helical domains upon membrane binding. We show that ATP binding enables partial insertion of EHD2 into the membrane, where G-domain-mediated oligomerization occurs. ATP hydrolysis is related to detachment of EHD2 from the membrane. Finally, we demonstrate that the regulation of EHD2 oligomerization in a membrane-bound state is crucial to restrict caveolae dynamics in cells.
EH 结构域蛋白 2（EHD2）是一种与动力蛋白相关的 ATP 酶，通过限制这些质膜凹陷的断裂和随后的内吞作用，将质膜窖限制在细胞膜表面。为此，EHD2 首先被认为与膜结合，然后寡聚化，最后在严格调控的机制循环中脱落。目前尚不清楚在此过程中如何使用 ATP，以及膜结合是否与蛋白构象变化偶联。在这里，我们表明，调节 N 端残基和 EH 结构域使 EHD2 二聚体在溶液中处于自动抑制构象。通过显著推进红外反射吸收光谱的应用，我们证明 EHD2 在与膜结合时通过倾斜螺旋结构域采用开放构象。我们表明，ATP 结合使 EHD2 部分插入膜中，在此处 G 结构域介导寡聚化发生。ATP 水解与 EHD2 从膜上的脱离有关。最后，我们证明在膜结合状态下对 EHD2 寡聚化的调节对于限制细胞中质膜窖的动力学至关重要。
