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bioRxiv · 10.1101/2023.12.07.570580

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

Larsen, S. T.·Dannersoe, J. K.·Nielsen, C. J. F.·Poulsen, L. R.·Palmgren, M.·Nissen, P.
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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, {Delta}20, {Delta}30, {Delta}35, {Delta}37, {Delta}40, {Delta}74 and {Delta}100) to explore the role of conserved motifs in the N-terminal segment preceding the calmodulin binding sites. Furthermore, we purified WT, {Delta}20- and {Delta}100-ACA8, tested activity in vitro and performed structural studies of purified {Delta}20-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 [A] 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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Larsen, S. T., Dannersoe, J. K., Nielsen, C. J. F., Poulsen, L. R., Palmgren, M., Nissen, P.. 2023-12-08. Conserved N-terminal Regulation of the ACA8 Calcium Pump with Two Calmodulin Binding Sites. https://doi.org/10.1101/2023.12.07.570580

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