Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ, 85721, USA.
Department of Biochemistry and Biophysics, University of California - San Francisco, San Francisco, CA, 94158, USA.
Nat Commun. 2022 Mar 11;13(1):1278. doi: 10.1038/s41467-022-28811-w.
Yeast Cadmium Factor 1 (Ycf1) sequesters heavy metals and glutathione into the vacuole to counter cell stress. Ycf1 belongs to the ATP binding cassette C-subfamily (ABCC) of transporters, many of which are regulated by phosphorylation on intrinsically-disordered domains. The regulatory mechanism of phosphorylation is still poorly understood. Here, we report two cryo-EM structures of Ycf1 at 3.4 Å and 4.0 Å resolution in inward-facing open conformations that capture previously unobserved ordered states of the intrinsically disordered regulatory domain (R-domain). R-domain phosphorylation is clearly evident and induces a topology promoting electrostatic and hydrophobic interactions with Nucleotide Binding Domain 1 (NBD1) and the Lasso motif. These interactions stay constant between the structures and are related by rigid body movements of the NBD1/R-domain complex. Biochemical data further show R-domain phosphorylation reorganizes the Ycf1 architecture and is required for maximal ATPase activity. Together, we provide insights into how R-domains control ABCC transporter activity.
酵母镉因子 1（Ycf1）将重金属和谷胱甘肽隔离到液泡中，以抵抗细胞应激。Ycf1 属于 ABC 转运蛋白 C 亚家族（ABCC）的转运蛋白，其中许多转运蛋白受固有无序结构域磷酸化的调节。磷酸化的调节机制仍知之甚少。在这里，我们报告了 Ycf1 的两个冷冻电镜结构，分辨率分别为 3.4Å 和 4.0Å，处于内向开放构象，捕获了先前未观察到的固有无序调节域（R 域）的有序状态。R 域磷酸化清晰可见，并诱导与核苷酸结合域 1（NBD1）和套索基序的拓扑结构促进静电和疏水相互作用。这些相互作用在结构之间保持不变，并通过 NBD1/R 域复合物的刚体运动相关。生化数据进一步表明，R 域磷酸化重组了 Ycf1 的结构，并且是最大 ATP 酶活性所必需的。总之，我们提供了关于 R 域如何控制 ABCC 转运蛋白活性的见解。
