{"title":"具有两个钙调蛋白结合位点的 ACA8 钙泵 N 端保守调控。","authors":"","doi":"10.1016/j.jmb.2024.168747","DOIUrl":null,"url":null,"abstract":"<div><p>The autoinhibited plasma membrane calcium ATPase ACA8 from <em>A. thaliana</em> 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.</p><p>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 <em>in vitro</em> and performed structural studies of purified Δ20-ACA8 stabilized in a lipid nanodisc to explore the mechanism of autoinhibition.</p><p>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.</p></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":null,"pages":null},"PeriodicalIF":4.7000,"publicationDate":"2024-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S0022283624003565/pdfft?md5=b8cc8fcfc2d15016e9e97d1a6090aedc&pid=1-s2.0-S0022283624003565-main.pdf","citationCount":"0","resultStr":"{\"title\":\"Conserved N-terminal Regulation of the ACA8 Calcium Pump with Two Calmodulin Binding Sites\",\"authors\":\"\",\"doi\":\"10.1016/j.jmb.2024.168747\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p>The autoinhibited plasma membrane calcium ATPase ACA8 from <em>A. thaliana</em> 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.</p><p>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 <em>in vitro</em> and performed structural studies of purified Δ20-ACA8 stabilized in a lipid nanodisc to explore the mechanism of autoinhibition.</p><p>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.</p></div>\",\"PeriodicalId\":369,\"journal\":{\"name\":\"Journal of Molecular Biology\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":4.7000,\"publicationDate\":\"2024-08-20\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.sciencedirect.com/science/article/pii/S0022283624003565/pdfft?md5=b8cc8fcfc2d15016e9e97d1a6090aedc&pid=1-s2.0-S0022283624003565-main.pdf\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Journal of Molecular Biology\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S0022283624003565\",\"RegionNum\":2,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"BIOCHEMISTRY & MOLECULAR BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of Molecular Biology","FirstCategoryId":"99","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0022283624003565","RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
引用次数: 0
摘要
拟南芥质膜钙离子ATP酶ACA8具有一个N末端的自动抑制结构域。钙载钙调蛋白与位于 42-62 和 74-96 残基的两个位点的结合可解除对 ACA8 活性的自动抑制。通过活性研究和酵母互补试验，我们研究了野生型（WT）和 N 端截短的 ACA8 构建物（Δ20、Δ30、Δ35、Δ37、Δ40、Δ74 和 Δ100），以探索钙调蛋白结合位点之前的 N 端片段中保守基团的作用。此外，我们纯化了WT、Δ20和Δ100-ACA8，在体外测试了其活性，并对稳定在脂质纳米盘中的纯化Δ20-ACA8进行了结构研究，以探索其自动抑制机制。我们的研究表明，残基 20 和 35 之间的 N 端片段（包括钙调蛋白结合位点上游的保守 Phe32）对于钙调蛋白的自动抑制和激活非常重要。以 3.3 Å 分辨率测定的氟化铍抑制 E2 状态的低温电子显微镜结构和低分辨率测定的 E1 状态的低温电子显微镜结构与 AlphaFold 预测相结合，提供了一个与突变研究相一致的自动抑制模型。
Conserved N-terminal Regulation of the ACA8 Calcium Pump with Two Calmodulin Binding Sites
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.
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