Conserved N-terminal Regulation of the ACA8 Calcium Pump with Two Calmodulin Binding Sites
biorxiv(2023)
摘要
The autoinhibited plasma membrane calcium ATPase, ACA8 from A. thaliana has an N-terminal autoinhibitory domain. Calcium-bound calmodulin binding at two sites located at residues 42-62 and 74-96 relieves autoinhibition of ACA8 activity.
We investigated N-terminally truncated ACA8 constructs (WT, Δ20, Δ30, Δ35, Δ37, Δ40, Δ74 and Δ100) to explore the role of conserved motifs in the N-terminal segment preceding the calmodulin binding sites. Furthermore, we purified WT, Δ20- and Δ100-ACA8, tested activity in vitro and performed structural studies of purified Δ20-ACA8 stabilized in its native form to explore the mechanism of autoinhibition.
Through activity studies and a yeast complementation assay, we show that an N-terminal segment between residues 20 and 35, upstream of the calmodulin binding sites, is important for autoinhibition and the activation by calmodulin, and that a conserved Phe32 is essential for autoinhibition. Cryo-EM structure determination at 3.3 Å resolution of a beryllium fluoride inhibited form shows no autoinhibition, but a low-resolution structure for an E1 state indicates autoinhibitory domain binding consistent with the mutational studies and AlphaFold predicted structures.
### Competing Interest Statement
The authors have declared no competing interest.
更多查看译文
AI 理解论文
溯源树
样例
![](https://originalfileserver.aminer.cn/sys/aminer/pubs/mrt_preview.jpeg)
生成溯源树,研究论文发展脉络
Chat Paper
正在生成论文摘要