https://ror.org/03m2x1q45 Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ, USA.
https://ror.org/03m2x1q45 Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ, USA
Life Sci Alliance. 2024 Aug 29;7(11). doi: 10.26508/lsa.202402779. Print 2024 Nov.
Many ATP-binding cassette transporters are regulated by phosphorylation on long and disordered loops which presents 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 3.23 Å cryo-EM structure reveals an R-domain structure with four phosphorylated residues and the position for the entire R-domain. The structure reveals key R-domain interactions including a bridging interaction between NBD1 and NBD2 and an interaction with the R-insertion, another regulatory region. We scanned these interactions by systematically replacing segments along the entire R-domain with scrambled combinations of alanine, glycine, and glutamine and probing function under cellular conditions that require the Ycf1 function. We find a close match with these interactions and interacting regions on our R-domain structure that points to the importance of most well-structured segments for function. We propose a model where the R-domain stabilizes a transport-competent state upon phosphorylation by enveloping NBD1 entirely.
许多 ATP 结合盒转运蛋白通过长而无序环上的磷酸化调节，这给结构方法的可视化带来了挑战。我们通过酶促富集磷酸化状态来捕获酵母镉因子 1（Ycf1）调节域（R 域）的激活状态。一个 3.23Å 的冷冻电镜结构揭示了一个带有四个磷酸化残基和整个 R 域位置的 R 域结构。该结构揭示了关键的 R 域相互作用，包括 NBD1 和 NBD2 之间的桥接相互作用以及与 R 插入物（另一个调节区域）的相互作用。我们通过系统地用丙氨酸、甘氨酸和谷氨酰胺的随机组合替换整个 R 域的片段，并在需要 Ycf1 功能的细胞条件下探测功能，扫描这些相互作用。我们在 R 域结构上找到了与这些相互作用和相互作用区域的紧密匹配，这表明大多数结构良好的片段对于功能非常重要。我们提出了一个模型，其中 R 域通过完全包围 NBD1 来稳定磷酸化后的运输能力状态。
