Conserved N-terminal Regulation of the ACA8 Calcium Pump with Two Calmodulin Binding Sites

Sigrid Thirup Larsen; Josephine Karlsen Dannersø; Christine Juul Fælled Nielsen; Lisbeth Rosager Poulsen; Michael Palmgren; Poul Nissen

doi:10.1016/j.jmb.2024.168747

Conserved N-terminal Regulation of the ACA8 Calcium Pump with Two Calmodulin Binding Sites

Sigrid Thirup Larsen, Josephine Karlsen Dannersø, Christine Juul Fælled Nielsen, Lisbeth Rosager Poulsen, Michael Palmgren, Poul Nissen^*

^*Corresponding author for this work

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaper › Journal article › Research › peer-review

Abstract

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.

Original language	English
Article number	168747
Journal	Journal of Molecular Biology
Volume	436
Issue	20
ISSN	0022-2836
DOIs	https://doi.org/10.1016/j.jmb.2024.168747
Publication status	Published - 15 Oct 2024

Keywords

autoinhibition
calcium ATPase
calmodulin
cryo-EM
N-terminal truncations

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@article{b933a53a907d4c2fa16acf923ba95a2c,

title = "Conserved N-terminal Regulation of the ACA8 Calcium Pump with Two Calmodulin Binding Sites",

abstract = "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 {\AA} 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.",

keywords = "autoinhibition, calcium ATPase, calmodulin, cryo-EM, N-terminal truncations",

author = "Larsen, {Sigrid Thirup} and Danners{\o}, {Josephine Karlsen} and Nielsen, {Christine Juul F{\ae}lled} and Poulsen, {Lisbeth Rosager} and Michael Palmgren and Poul Nissen",

note = "Publisher Copyright: {\textcopyright} 2024 The Author(s)",

year = "2024",

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day = "15",

doi = "10.1016/j.jmb.2024.168747",

language = "English",

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Conserved N-terminal Regulation of the ACA8 Calcium Pump with Two Calmodulin Binding Sites. / Larsen, Sigrid Thirup; Dannersø, Josephine Karlsen ; Nielsen, Christine Juul Fælled et al.
In: Journal of Molecular Biology, Vol. 436, No. 20, 168747, 15.10.2024.

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaper › Journal article › Research › peer-review

TY - JOUR

T1 - Conserved N-terminal Regulation of the ACA8 Calcium Pump with Two Calmodulin Binding Sites

AU - Larsen, Sigrid Thirup

AU - Dannersø, Josephine Karlsen

AU - Nielsen, Christine Juul Fælled

AU - Poulsen, Lisbeth Rosager

AU - Palmgren, Michael

AU - Nissen, Poul

PY - 2024/10/15

Y1 - 2024/10/15

N2 - 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.

AB - 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.

KW - autoinhibition

KW - calcium ATPase

KW - calmodulin

KW - cryo-EM

KW - N-terminal truncations

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DO - 10.1016/j.jmb.2024.168747

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C2 - 39168442

AN - SCOPUS:85202515295

SN - 0022-2836

VL - 436

JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

IS - 20

M1 - 168747

ER -

Conserved N-terminal Regulation of the ACA8 Calcium Pump with Two Calmodulin Binding Sites

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Keywords

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