Cryo-EM structure of the tetra-phosphorylated R-domain in Ycf1 reveals key interactions for transport regulation.
Cryo-EM structure of the tetra-phosphorylated R-domain in Ycf1 reveals key interactions for transport regulation.
复制标题
Ycf1 中四磷酸化 R 结构域的冷冻电镜结构揭示了运输调节的关键相互作用。
DOI：
10.1101/2024.03.06.583773
复制
发表时间：
2024
期刊：
影响因子：
--
通讯作者：
Tomasiak,ThomasM
中科院分区：
文献类型：
--
作者：
deCarvalho,RodolphoSA;Rasel,ShamiulI;Khandelwal,NiteshK;Tomasiak,ThomasM
许多ATP结合盒（ABC）转运蛋白是由长而无序的环上的磷酸化调控的，这对结构方法的可视化提出了挑战。我们通过酶促富集磷酸化状态捕获了酵母镉因子1（Ycf 1）的调节结构域（R-结构域）的活化状态。一个3.2 μ m的cryo-EM结构揭示了一个R-结构域的结构与四个磷酸化的残基和整个R-结构域的位置。该结构揭示了关键的R-结构域相互作用，包括NBD 1和NBD 2之间的桥接相互作用以及与R-插入（另一个调控区）的相互作用。我们系统地探测这些相互作用与连接器取代策略沿着的R-结构域，并找到一个紧密的匹配与这些相互作用和生存下Ycf 1依赖的生长条件。我们提出了一个模型，其中四个重叠的磷酸化位点桥接几个区域的Ycf 1从事运输能力的状态。
Many ATP-binding cassette (ABC) transporters are regulated by phosphorylation on long and disordered loops which present a challenge to visualize with structural methods. We have trapped an activated state of the regulatory domain (R-domain) of Yeast Cadmium Factor 1 (Ycf1) by enzymatically enriching the phosphorylated state. A 3.2 Å cryo-EM structure reveals an R-domain structure with four phosphorylated residues and a position for the entire R-domain. The structure reveals key R-domain interactions including a bridging interaction between NBD1 and NBD2 as well as an interaction with the R-insertion, another regulatory region. We systematically probe these interactions with a linker substitution strategy along the R-domain and find a close match with these interactions and survival under Ycf1-dependent growth conditions. We propose a model where four overlapping phosphorylation sites bridge several regions of Ycf1 to engage in a transport-competent state.