Conformational changes during the reaction cycle of Plasma Membrane Ca2+-ATPase in the autoinhibited and activated states

Saffioti, Nicolás A.; Sautu, Marilina de; Riesco, Ana Sol; Ferreira-Gomes, Mariela; Rossi, Juan Pablo F.C.; Mangialavori, Irene C.

doi:10.1042/bcj20210036

Cited by 8 publications

(9 citation statements)

References 60 publications

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“…In contrast, ATP8B1 remains poorly studied at the molecular mechanistic level. In particular, while several other P4-ATPases, and P-type ATPases in general, are tightly regulated by lipid co-factors, protein partners, or by their terminal extensions ( Azouaoui et al, 2017 ; Chalat et al, 2017 ; Holemans et al, 2015 ; Saffioti et al, 2021 ; Tsai et al, 2013 ), the way ATP8B1 activity is regulated remains unknown. Recent high-resolution structures of the yeast Drs2-Cdc50, Dnf1,2-Lem3 and the human ATP8A1-CDC50A and ATP11C-CDC50A flippase complexes have illuminated the molecular mechanism of lipid transport, providing a framework for understanding how these transporters are able to move lipids ( Bai et al, 2019 ; Bai et al, 2020 ; Hiraizumi et al, 2019 ; Lyons et al, 2020 ; Nakanishi et al, 2020b ; Timcenko et al, 2019 ; Timcenko et al, 2021 ).…”

Section: Introductionmentioning

confidence: 99%

Autoinhibition and regulation by phosphoinositides of ATP8B1, a human lipid flippase associated with intrahepatic cholestatic disorders

Dieudonné

Herrera

Laursen

et al. 2022

eLife

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P4-ATPases flip lipids from the exoplasmic to the cytosolic leaflet, thus maintaining lipid asymmetry in eukaryotic cell membranes. Mutations in several human P4-ATPase genes are associated with severe diseases, e.g. in ATP8B1 causing progressive familial intrahepatic cholestasis, a rare inherited disorder progressing toward liver failure. ATP8B1 forms a binary complex with CDC50A and displays a broad specificity to glycerophospholipids, but regulatory mechanisms are unknown. Here, we report functional studies and the cryo-EM structure of the human lipid flippase ATP8B1-CDC50A at 3.1 Å resolution. We find that ATP8B1 is autoinhibited by its N- and C-terminal tails, which form extensive interactions with the catalytic sites and flexible domain interfaces. Consistently, ATP hydrolysis is unleashed by truncation of the C-terminus, but also requires phosphoinositides, most markedly phosphatidylinositol-3,4,5-phosphate (PI(3,4,5)P3), and removal of both N- and C-termini results in full activation. Restored inhibition of ATP8B1 truncation constructs with a synthetic peptide mimicking the C-terminal segment further suggests molecular communication between N- and C-termini in the autoinhibition and demonstrates that the regulatory mechanism can be interfered with by exogenous compounds. A recurring (G/A)(Y/F)AFS motif of the C-terminal segment suggests that this mechanism is employed widely across P4-ATPase lipid flippases in plasma membrane and endomembranes.

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Section: Introductionmentioning

confidence: 99%

Autoinhibition and regulation by phosphoinositides of ATP8B1, a human lipid flippase associated with intrahepatic cholestatic disorders

Dieudonné

Herrera

Laursen

et al. 2022

eLife

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“…ACA8 shows the expected P-type ATPase features and is similar to the E2-BeFx complex of SERCA (Figure 2E). Unlike for P4-ATPases [43], however, the E2P state of ACA8 did not reveal any density for the autoinhibitory domain, and consistently, Saffioti et al also showed that the autoinhibitory interaction is disrupted in the E2P state [47].…”

Section: Discussionmentioning

confidence: 99%

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

Larsen,

Dannersø,

Nielsen

et al. 2023

Preprint

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The autoinhibited plasma membrane calcium ATPase, ACA8 fromA. thalianahas 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 activityin vitroand 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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“…PMCA belongs to the P-ATPases, a family of proteins that utilize the energy derived from ATP hydrolysis to transport ions and molecules across biological membranes 13 . Like all P-ATPases, PMCA follows the E1–E2 model and undergoes various conformational changes as part of its reaction cycle to carry out its transport function 14 . This model proposes that cytoplasmic Ca 2+ binds with high affinity to E 1 state, forming E 1Ca, which is then phosphorylated by ATP, leading to E 1(Ca)P. In E 1(Ca)P state, Ca 2+ is occluded and inaccessible to both sides of the membrane.…”

Section: Introductionmentioning

confidence: 99%

Magnesium enhances aurintricarboxylic acid’s inhibitory action on the plasma membrane Ca2+-ATPase

Souto-Guevara,

Obiol,

Bruno

et al. 2024

Sci Rep

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Our research aimed to elucidate the mechanism by which aurintricarboxylic acid (ATA) inhibits plasma membrane Ca2+-ATPase (PMCA), a crucial enzyme responsible for calcium transport. Given the pivotal role of PMCA in cellular calcium homeostasis, understanding how it is inhibited by ATA holds significant implications for potentially regulating physiopathological cellular processes in which this pump is involved. Our experimental findings revealed that ATA employs multiple modes of action to inhibit PMCA activity, which are influenced by ATP but also by the presence of calcium and magnesium ions. Specifically, magnesium appears to enhance this inhibitory effect. Our experimental and in-silico results suggest that, unlike those reported in other proteins, ATA complexed with magnesium (ATA·Mg) is the molecule that inhibits PMCA. In summary, our study presents a novel perspective and establishes a solid foundation for future research efforts aimed at the development of new pharmacological molecules both for PMCA and other proteins.

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Conformational changes during the reaction cycle of Plasma Membrane Ca2+-ATPase in the autoinhibited and activated states

Cited by 8 publications

References 60 publications

Autoinhibition and regulation by phosphoinositides of ATP8B1, a human lipid flippase associated with intrahepatic cholestatic disorders

Autoinhibition and regulation by phosphoinositides of ATP8B1, a human lipid flippase associated with intrahepatic cholestatic disorders

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

Magnesium enhances aurintricarboxylic acid’s inhibitory action on the plasma membrane Ca2+-ATPase

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