Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark; Danish Research Institute of Translational Neuroscience - DANDRITE, Aarhus University, Aarhus, Denmark.
Department of Plant and Environmental Sciences, Copenhagen University, Thorvaldsensvej 40, DK-1871, Denmark.
J Mol Biol. 2024 Oct 15;436(20):168747. doi: 10.1016/j.jmb.2024.168747. Epub 2024 Aug 20.
The autoinhibited plasma membrane calcium ATPase ACA8 from A. thaliana has an N-terminal autoinhibitory domain. Binding of calcium-loaded calmodulin at two sites located at residues 42-62 and 74-96 relieves autoinhibition of ACA8 activity. Through activity studies and a yeast complementation assay we investigated wild-type (WT) and N-terminally truncated ACA8 constructs (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 a lipid nanodisc to explore the mechanism of autoinhibition. We show that an N-terminal segment between residues 20 and 35 including conserved Phe32, upstream of the calmodulin binding sites, is important for autoinhibition and the activation by calmodulin. Cryo-EM structure determination at 3.3 Å resolution of a beryllium fluoride inhibited E2 form, and at low resolution for an E1 state combined with AlphaFold prediction provide a model for autoinhibition, consistent with the mutational studies.
拟南芥中的自抑制质膜钙 ATP 酶 ACA8 具有 N 端自抑制结构域。位于残基 42-62 和 74-96 处的两个钙结合钙调蛋白结合位点可解除 ACA8 活性的自抑制。通过活性研究和酵母互补测定，我们研究了野生型（WT）和 N 端截断的 ACA8 构建体（Δ20、Δ30、Δ35、Δ37、Δ40、Δ74 和 Δ100），以探讨钙调蛋白结合位点之前的 N 端片段中保守基序的作用。此外，我们纯化了 WT、Δ20-和 Δ100-ACA8，在体外测试了它们的活性，并对稳定在脂质纳米盘中的纯化 Δ20-ACA8 进行了结构研究，以探讨自抑制的机制。我们表明，位于钙调蛋白结合位点上游的残基 20-35 之间的 N 端片段（包括保守的 Phe32）对于自抑制和钙调蛋白的激活很重要。使用铍氟化物抑制的 E2 形式的 3.3Å 分辨率冷冻电镜结构测定，以及与 AlphaFold 预测相结合的低分辨率 E1 状态，提供了一个自抑制模型，与突变研究一致。
