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Conserved N-terminal Regulation of the ACA8 Calcium Pump with Two Calmodulin Binding Sites
Open accessPreprint — published as 10.1016/j.jmb.2024.168747
Abstract
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.
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Metadata
- Authors
- Larsen, S. T., Dannersø, J. K., Nielsen, C. J. F., Poulsen, L. R., Palmgren, M., & Nissen, P.
- Affiliations
- Aarhus University (DK)
- University of Copenhagen (DK)
- Corresponding author
- Nissen, Poul
- ORCID
- Venue
- Journal of Molecular Biology
- Publisher
- openRxiv
- Published
- 2023-12-08
- Volume
- 436
- Issue
- 20
- Pages
- 168747-168747
- ISSN
- 0022-2836 (ISSN-L)
- OpenAlex
- W4401759289
- Cited by
- 6
- References
- 1
- Citations by year
- 2026: 3 · 2025: 3
- FWCI
- 0.82
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- cc-by
- Locations
- Journal of Molecular Biology (publishedVersion, cc-by)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
- PubMed (publishedVersion)
- Research at the University of Copenhagen (University of Copenhagen) (publishedVersion, cc-by)
- Funders
- Natur og Univers, Det Frie Forskningsråd (7014-00328B)
- National Research Foundation
- Danmarks Grundforskningsfond
- Lundbeckfonden (R248‐2016‐2518, 2016-2518, DANDRITE‐R248‐2016‐2518, R310‐2018‐3713, R155-2015-2666)
- H. Lundbeck A/S
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › ATP Synthase and ATPases Research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ion channel regulation and function
- Keywords
- plasma membrane calcium ATPase, calmodulin binding site, autoinhibition, Arabidopsis thaliana, calmodulin activation, lipid nanodiscs, cryo-EM structure, yeast complementation, N-terminal truncation
- MeSH terms
- Calcium-Transporting ATPases, Beryllium, Binding Sites, Calcium, Calmodulin, Fluorides, Models, Molecular, Protein Binding, Protein Conformation, Arabidopsis, Cryoelectron Microscopy, Arabidopsis Proteins
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