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TitleConserved N-terminal Regulation of the ACA8 Calcium Pump with Two Calmodulin Binding Sites.
Journal, issue, pagesJ Mol Biol, Vol. 436, Issue 20, Page 168747, Year 2024
Publish dateOct 15, 2024
AuthorsSigrid Thirup Larsen / Josephine Karlsen Dannersø / Christine Juul Fælled Nielsen / Lisbeth Rosager Poulsen / Michael Palmgren / Poul Nissen /
PubMed AbstractThe 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 ...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.
External linksJ Mol Biol / PubMed:39168442
MethodsEM (single particle)
Resolution3.3 Å
Structure data

18506

8qmp

EMDB-18506, PDB-8qmp:
Structure of the E2 Beryllium Fluoride Complex of the Autoinhibited Calcium ATPase ACA8
Method: EM (single particle) / Resolution: 3.3 Å

Chemicals

ChemComp-MG:
Unknown entry

ChemComp-BEF:
BERYLLIUM TRIFLUORIDE ION

Source
  • arabidopsis thaliana (thale cress)
KeywordsTRANSPORT PROTEIN / HYDROLASE Calcium transporter P-type ATPase / HYDROLASE

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