Mechanistic insights into autoinhibition of the human flippase ATP8B1

Mechanistic insights into autoinhibition of the human flippase ATP8B1 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Mechanistic insights into autoinhibition of the human flippase ATP8B1 Michelle Juknaviciute Laursen, Mathilde Roth, Poul Nissen, Charlott Stock, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9243799/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 9 You are reading this latest preprint version Abstract P4-ATPases are lipid flippases that maintain membrane phospholipid asymmetry by transporting specific phospholipids from the exoplasmic to the cytosolic leaflet, an essential process for membrane integrity, trafficking and signaling. Several P4-ATPases are tightly regulated by autoinhibitory N- and C-terminal extensions, yet the molecular basis of this regulation remains incompletely understood. Here, we investigated the autoinhibition mechanism of the human flippase ATP8B1 using trans-inhibition assays with synthetic peptides derived from its C-terminal tail. Using purified C-terminally truncated ATP8B1-CDC50A, we systematically assessed the inhibitory properties of peptides corresponding to distinct segments of the C-terminus. We show that the distal disordered region of the C-terminal tail significantly contributes to autoinhibition, likely through transient interactions with the cytosolic domains. We further identify a critical interaction between R1228 in the C-terminal tail and E219 in the A-domain, whose disruption markedly reduces inhibitory potency. In addition, we demonstrate that a minimal peptide spanning residues 1216–1228, which bridges the A- and N-domains in the autoinhibited conformation, is sufficient to inhibit ATPase activity. Together, these results refine the molecular description of ATP8B1 autoinhibition, open the way for structure-based activation strategies and provide insight into conserved regulatory mechanisms among P4-ATPases. P-type ATPase Flippases P4-ATPase Autoinhibition ATP8B1 PFIC1 Intrahepatic cholestasis Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Membranes of the late secretory pathway of eukaryotes are characterized by an asymmetric distribution of phospholipids. This asymmetry is defined, but not limited, by an enrichment of sphingolipids and phosphatidylcholine (PtdCho) in the exoplasmic leaflet, whereas phosphatidylserine (PtdSer), phosphatidylethanolamine (PtdEth) are mostly found in the cytosolic leaflet (Caputo et al., 2023). This asymmetry is essential for numerous cellular processes, in particular signaling and membrane trafficking events, where the enrichment of PtdSer on the cytoplasmic leaflet of the plasma membrane mediates the specific recruitment of peripheral proteins such as RAS small GTPases (Sakuragi & Nagata, 2023 , Leventis & Grinstein, 2010 ). P4-ATPases, also known as flippases, participate in the establishment and maintenance of this asymmetry by transporting specific phospholipids from the exoplasmic to the cytosolic leaflet of membranes, in an ATP-dependent manner (Sakuragi & Nagata, 2023 ). P4-ATPases are part of the P-type ATPase superfamily and therefore share a common architecture, with an α-helical transmembrane domain containing the lipid transport site, connected to three cytosolic domains: the nucleotide-binding (N) domain, which binds ATP; the phosphorylation (P) domain, which contains a conserved aspartate residue that is transiently phosphorylated during the transport cycle; and the actuator (A) domain, responsible for transporter dephosphorylation and return of the enzyme to its initial state (Fig. 1 ) (Palmgren, 2023 , Stock et al., 2023 ). In addition, most P4-ATPases form a binary complex with a subunit from the CDC50 family, which mediates their proper function and localization (Bryde et al., 2010 ). The structural details of the lipid transport cycle of P4-type ATPases have been extensively studied by cryoEM and X-ray crystallography and follow the Post-Albers model, with two main phases: the E1 phase, in which the P4-ATPase undergoes phosphorylation in a lipid-independent manner, and the E2 phase, in which lipid binding and occlusion within the protein trigger dephosphorylation of the conserved catalytic aspartate in the P-domain by an invariant glutamate of the A-domain (Sai & Lee, 2024 ; Duan & Li, 2024 ). Several P4-ATPases have been shown to be tightly regulated by their N- and/or C-terminal extensions. In particular, the yeast flippase Drs2-Cdc50 and the human flippases ATP8A2-CDC50A and ATP8B1-CDC50A have been reported to be autoinhibited through their terminal extensions (Zhou et al, 2013 ; Azouaoui et al., 2017 ; Matsell et al., 2025 ; Dieudonné et al., 2022 ). CryoEM structures of the Drs2-Cdc50 and ATP8B1-CDC50A complexes in both autoinhibited and active conformations revealed that the C-terminal tail of these P4-ATPases can establish extensive interactions with the three cytosolic domains of the enzyme after ATP hydrolysis, thereby locking the transporter in E2P autoinhibited conformations (Timcenko et al., 2019 ; Timcenko et al., 2021 ; Cheng et al., 2022 ; Dieudonné et al., 2022 ; Dieudonné et al., 2023 ). In this state, the P4-ATPase is unable to occlude its substrate lipid, a step that is required for lipid translocation. In ATP8B1, this regulatory mechanism is further reinforced by the N-terminal tail, which inserts between the A and P domains once the C-terminal tail is engaged, further restricting the cytosolic domain rearrangements required to proceed through the catalytic lipid transport cycle (Fig. 1 B) (Dieudonné et al., 2022 ). Mutations or deletion of this C-terminal tail region in ATP8A2 or Drs2 have been shown to have a drastic effect on protein expression level, likely due to protein instability (Azouaoui et al., 2017 , Matsell et al., 2025 ). Inherited mutations in P4-ATPases have been associated with various human diseases (Shin and Takatsu, 2025 ). Well-documented examples include progressive familial intrahepatic cholestasis (PFIC) type 1 and the less severe benign recurrent intrahepatic cholestasis (BRIC1), which both result directly from mutations in ATP8B1 (Klomp et al, 2004 ). While previous studies have investigated ATP8B1 lipid transport specificity and function (Muranaka et al, 2024 ; Dieudonné et al., 2023 ), very few studies have explored the regulatory mechanisms of ATP8B1, in particular the role of the different regions of its C-terminal tail in the autoinhibition process. Previously, our group used purified ATP8B1-CDC50A lacking its C-terminal tail to perform trans-inhibition assays with synthetic peptides to investigate the role of the N- and C-terminal tails of ATP8B1, as well as the putative role of unknown kinase(s) in the activation mechanism of ATP8B1 by phosphorylation of S1223 (Dieudonné et al., 2022 ). Here, we used a similar approach with peptides corresponding to fragments of the ATP8B1 C-terminal tail to gain further insight into the role of the different sections of this autoinhibitory region of the flippase. This approach allowed us to define the contribution of specific segments, providing a clearer description of the autoinhibition mechanism of ATP8B1 and, more broadly, of other flippases regulated in a similar manner. Methods Peptides All Peptides presented is this study were synthetized by Biomatik Company (Canada) with > 90% purity grade, TFA free. The lyophilized peptides were directly resuspended in buffer A (50mM MOPS-Tris pH 7, 100 mM KCl) and stored at -20°C before use. Heterologous co-expression and purification of the ATP8B1-CDC50A complex Yeast cultures, recombinant protein expression and membrane preparation were performed as described previously (Azouaoui et al., 2016 , Dieudonné et al., 2023 ). Purification of C-terminal cleavable hATP8B1(HRV 3C protease L1185)-hCDC50A complex was performed as described previously (Dieudonné et al., 2023 ). The final protein concentration of three independent biological triplicates was determined by loading duplicate samples from each purification on a Coomassie blue-stained SDS-PAGE, with purified Drs2-Cdc50 complex of known amount (determined by absorbance at 280 nm). ATPase activity assay To estimate the effect of treating C-terminally truncated ATP8B1 (Δ1185) - CDC50A complex with different peptides, ATP hydrolysis was measured using an enzyme-coupled assay (Sehgal et al., 2016 ). To measure the ATPase activity of C-terminally truncated ATP8B1 (Δ1185), samples were prepared containing 0.75 µg.mL⁻¹ ATP8B1-CDC50A, 0.15 mg.mL⁻¹ PtdEth, 0.025 mg.mL⁻¹ PI(3,4,5)P 3 , 0.04 mg.mL⁻¹ PK, 0.1 mg.mL⁻¹ LDH, 1 mM PEP, 1 mg.mL⁻¹ DDM, and 0.2 mg.mL⁻¹ CHS in buffer A without glycerol. To allow proper lipid diffusion within flippase-containing detergent micelles, samples were incubated for 1 h 30 min at 4°C prior to the addition of 165 µM NADH. ATPase activity assays were performed in 96-well plates, with 350 µL sample per well. The decrease in absorbance at 340 nm was monitored for 8 min per run at 37°C using a SpectraMax® i3 microplate reader in kinetic mode. After an initial run for background measurement, ATP was added to a final concentration of 1 mM to measure ATPase activity prior to peptide addition. This was followed by up to three additional runs with increasing peptide concentrations. To assess peptide effects on ATPase activity, the activity measured in each run was normalized to the activity of the same sample before peptide addition, after background correction. Each peptide was tested at nine different concentrations ranging from 1 nM to 6.6 µM. Technical triplicates were performed for each concentration and for three independent biological replicates of purified C-terminally truncated ATP8B1 (Δ1185)-CDC50A, resulting in a total of nine measurements per peptide concentration. Statistics IC 50 of each peptide was estimated, when possible, from the 9 replicates of 9 different peptide concentrations. For each peptide, dose-response nonlinear regression curve fits, as well as the associated 99% confidence band, were generated using GraphPad Prism software based on the associated covariance matrix. For the non-linear fit, the top was constrained to the normalized activity of 1. The non-linear fits are shown in solid, and the 99% confidence bands are shown as transparent bands enclosed in dashed lines. Results and Discussion The distal disordered region of the C-terminal tail of ATP8B1 contributes to the autoinhibition mechanism To gain insight into the structural basis of ATP8B1 autoinhibition mediated by its C-terminal region, we designed a series of peptides corresponding to different segments of the ATP8B1 C-terminal tail, named according to their position within ATP8B1 (Fig. 2 A-B). We then performed trans-inhibition assays of the ATPase activity of C-terminally truncated ATP8B1 (Δ1185) in detergent in the presence of the lipid substrate PtdEth and the activating lipid phosphatidylinositol trisphosphate (PI(3,4,5)P₃), and evaluated their inhibitory properties by determining IC 50 values (Table 1 ). As previously observed, the peptide spanning residues 1205–1251, corresponding to the full ATP8B1 C-terminal tail, efficiently inhibited ATP8B1 ATPase activity, with an IC 50 of 16 nM (Fig. 2 C; Table 1 ) (Dieudonné et al., 2022 ). Truncation of the last eight residues (1205–1243) moderately reduced inhibitory potency, yielding an IC 50 of 56 nM (Fig. 2 C; Table 1 ). In contrast, removal of the region corresponding to the disordered segment of the C-terminal tail unresolved in ATP8B1 cryoEM structures (1205–1228) resulted in an approximately tenfold increase in IC 50 to 175 nM, relative to full-length peptide 1205–1251. These results indicate that the disordered portion of the ATP8B1 C-terminal tail contributes to autoinhibition, likely through transient interactions with the cytosolic domains. Notably, ATP8A1 and ATP8A2 in contrast to ATP8B1 possess shorter C-terminal tails with smaller disordered distal segments. In the autoinhibited cryoEM structure of ATP8A1, only the last nine residues could not be modeled (Hiraizumi et al., 2019 ), compared with twenty-three residues in ATP8B1 (Dieudonné et al., 2022 ). Compared to ATP8B1 and the yeast homolog Drs2, ATP8A1 and ATP8A2 do show ATPase activity for the full-length proteins (Coleman et al., 2012 ; Hiraizumi et al., 2019 ; Matsell et al., 2024; Azouaoui et al., 2016 ; Dieudonné et al., 2022 ). The E243-R1228 interaction is critical for ATP8B1 autoinhibition The most distal region of the ATP8B1 C-terminus observed in the cryoEM structures interacts directly with the A-domain through a salt bridge (E219-R1228) (Fig. 3 A) and with the A- and N-domain through Van-der-Walls interactions in a hydrophobic patch formed by Y225, F239, M538, I593, I1225 and I1227 (Fig. 3 B). Hence, we evaluated the inhibitory properties of shorter peptides lacking either R1228 (1205–1227) or R1228 and I1227 (1205–1226) (Fig. 3 C). Removal of R1228 resulted in a fivefold increase in IC 50 , from 175 nM for peptide 1205–1228 to 907 nM for peptide 1205–1227 while the additional removal of I1227 results in an almost complete loss of the inhibition properties of the corresponding 1205–1226 peptide (Fig. 3 D; Table 1 ). To further assess the contribution of the E219-R1228 interaction, we tested the full-length C-terminal peptide in which R1228 was substituted with alanine (1205–1251 R1228A). This mutant peptide exhibited a 19-fold increase in IC 50 (305 nM) compared to the WT 1205–1251 peptide, indicating a major contribution of this residue to the autoinhibition (Fig. 3 E). However, despite the reduced potency of the R1228A mutant peptide, it retained a stronger inhibitory activity than peptide 1205–1227, with an IC 50 of 307 nM versus 907 nM, respectively, suggesting that residues located C-terminally to R1228 may partially compensate for the loss of this interaction. In particular, K1229, K1230, and R1231 could potentially engage in electrostatic interactions with E219, consistent with the apparent flexibility of this region of the C-terminal tail. Interestingly, structural alignment of the C-terminal regions of ATP8B1, ATP8A1, and Drs2 shows that R1228 in ATP8B1 is not conserved (Figure S1). Instead, in ATP8A1 and Drs2, which exhibit a higher degree of sequence and structural conservation with each other, the interaction with the conserved glutamate in the A-domain (E219 in ATP8B1) appears to be mediated by a hydrogen bond involving a conserved tyrosine residue (Y1292 and Y1139 for Drs2 and ATP8A1, respectively). Furthermore, the 1205–1251 peptide corresponding to the ATP8B1 C-terminal tail does not inhibit Drs2 ATPase activity (Dieudonné et al., 2022 ). In contrast to R1228, E219 is highly conserved among P4-ATPases, and not only in those displaying a long C-terminal tail harbouring a (G/A)(Y/F)AFS motif. In E1 states, it mediates intra-A-domain interactions of ATP8A1, Drs2 and even ATP11C. The 1216–1228 C-terminal region bridging the A- and N- domains is sufficient for autoinhibition Finally, we wanted to investigate the role of the highly conserved (G/A)(Y/F)AFS motif, known to interact with the ATP binding site of the N-domain in the E2P autoinhibited conformation. To this end, we designed two peptides: one spanning residues 1205–1215, containing the conserved motif along with additional polar and positively charged residues to ensure peptide solubility, and a second peptide corresponding to residues 1216–1228, corresponding to the region of the C-terminal tail of ATP8B1 intercalated in between the N- and A-domains (Fig. 4 A). The 1205–1215 peptide did not inhibit ATPase activity, whereas peptide 1216–1228 retained inhibitory properties with an IC 50 of 445 nM. Although the isolated (G/A)(Y/F)AFS conserved motif (1205–1215) was not sufficient to directly inhibit ATPase activity, its presence markedly enhanced inhibition in the context of the longer peptide (1205–1228) (Fig. 4 B, Table 1 ). Consistently, peptide 1205–1228 exhibited a substantially lower IC 50 of 147 nM, approximately three-fold lower than that of peptide 1216–1228. Together, these results indicate that the conserved (G/A)(Y/F)AFS motif is not sufficient for inhibition on its own but enhances the inhibitory capacity of the adjacent C-terminal region that intercalates the N- and A-domains. These results are also in line with previous work done on the yeast flippase Drs2 where truncation of its C-terminal tail by limited proteolysis was used to relieve the autoinhibition mechanism in vitro (Azouaoui et al., 2017 ). Indeed, the truncation by limited proteolysis of Drs2 leaves the (G/A)(Y/F)AFS motif while removing the last 64 residues of the Drs2 C-terminal tail, and results in an active flippase consuming ATP in a lipid-dependent manner. Conclusion From a structural perspective, it has been shown that autoinhibited ATP8B1 is locked in an E2P-like conformation, with a possible equilibrium between states with an open or closed lipid-binding site (Dieudonné et al., 2023 ). Based on this observation, we and others have proposed that C-terminal mediated autoinhibition restricts the rotation of the A-domain, a movement that normally results from structural rearrangements of TM1 and TM2 induced by lipid head-group binding prior to its occlusion within the transport site. Here, our data shows that the minimal region of the ATP8B1 C-terminal tail responsible for autoinhibition corresponds to the segment that most strongly interacts with the A domain, in line with that model. More importantly, the A-domain rotation that is blocked by the inhibitory peptide represents a conformational transition, which is highly conserved among P-type ATPases and directly linked to the dephosphorylation of the catalytic aspartate in the P-domain during the transport cycle. Given the strong conservation of this mechanistic step across the P-type ATPase superfamily, our results may have broader implications beyond ATP8B1. In particular, the identification of a minimal peptide segment capable of restricting A-domain motion suggests a potential strategy for the design of inhibitory peptides targeting other P-type ATPases. Conversely, a detailed understanding of this autoinhibitory interaction could also guide the rational design of molecules aimed at antagonizing C-terminal-mediated autoinhibition in P4-ATPases, thereby promoting enzyme activation. Finally, although our results provide important insights into the autoinhibition mechanism of ATP8B1 and P4-ATPases more broadly, how this autoinhibition is relieved in vivo remains to be determined. Table 1 Statistical analysis of the inhibition properties of the peptides mimicking different regions of the C-terminal tail of ATP8B1. ND: Not determined. Declarations Competing Interests The authors have no relevant financial or non-financial interests to disclose. Funding acquisition - PN TD Author Contribution Conceptualization - PN TDMethodology - CS TDInvestigation - MJL MRFormal analysis - MJL CS TDWriting original draft - MJLWriting review and editing - MR PN CS TDVisualization - MJL MR CS TDSupervision / project administration - PN CS TDFunding acquisition - PN TD Acknowledgement We thank Joseph Lyons, Amelie Benfeldt Purup, Filip Pamula, Line Marie Christiansen, Guillaume Lenoir, Cédric Montigny, and Poul Sørensen for fruitful discussions and insightful feedback throughout this project. We are also grateful to Anna Marie Nielsen and Karen Bech-Pedersen for their excellent technical and administrative assistance. Data Availability The data used to support the findings of this study are available from the corresponding authors upon request. 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J Biol Chem 288(44):31807–31815. https://doi.org/10.1074/jbc.M113.481986 Additional Declarations No competing interests reported. Supplementary Files floatimage6.png Figure S1: Structural alignment of ATP8B1, ATP8A1 and Drs2 C-terminal tail regions. (A) Structural alignment of the most distal part of the C-terminal tails of ATP8B1 (green), ATP8A1 (light blue), and Drs2 (dark blue) as resolved in their respective cryoEM structures. ATP8B1 (PDB: 7PY4), ATP8A1 (PDB: 6K7L), and Drs2 (PDB: 6ROH) were aligned based on their N-domains. (B) Corresponding sequence alignment showing the Cα distances measured in ChimeraX. The two hydrophobic residues of the hydrophobic patch are underlined with a *. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9243799","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":616002414,"identity":"f09d8bc6-17c3-4b77-960e-e922c6359daf","order_by":0,"name":"Michelle Juknaviciute Laursen","email":"","orcid":"","institution":"Aarhus University","correspondingAuthor":false,"prefix":"","firstName":"Michelle","middleName":"Juknaviciute","lastName":"Laursen","suffix":""},{"id":616002416,"identity":"cf545f29-2e24-41ca-adb3-8c420b0875a9","order_by":1,"name":"Mathilde Roth","email":"","orcid":"","institution":"University of Paris-Saclay","correspondingAuthor":false,"prefix":"","firstName":"Mathilde","middleName":"","lastName":"Roth","suffix":""},{"id":616002419,"identity":"ad8b9b2e-e2fc-43ce-9f5f-4c0578ad25c1","order_by":2,"name":"Poul Nissen","email":"","orcid":"","institution":"Aarhus University","correspondingAuthor":false,"prefix":"","firstName":"Poul","middleName":"","lastName":"Nissen","suffix":""},{"id":616002421,"identity":"0b917689-5258-480e-b723-a146377cb4ff","order_by":3,"name":"Charlott Stock","email":"","orcid":"","institution":"Aarhus University","correspondingAuthor":false,"prefix":"","firstName":"Charlott","middleName":"","lastName":"Stock","suffix":""},{"id":616002423,"identity":"1fd69e2f-0823-4b51-851d-7180c31b8d20","order_by":4,"name":"Thibaud Dieudonné","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABcUlEQVRIie2QMWvCQBiGLwQuHaJZL0T0LyQECsXS/pUcQl1EBKEIFXoSOKfSNaDQv2D3QhMOdDnsamkHRUgXB6dCaYVeYquJduhYSp7hu+QLD+97ASAj4y+CJCqmDAAThxptlE5nvv4mkfjIR6O1MdR9RQ1cO6XAaPCEEm+E4n8ryIFGqsiOcmq4dPpGyyA/kodo0Xqua/qcGI02qGs95i7Bx0kJKtwEwd0mpRB0rStaBTqDZ3qfh029h4ntDUETjTEVtSsWVWsm8MNtMbHPUQZMph4a4gEPnjCp5AjAhEtUXhHZgaBmyks/qeirWNFeY+X+MSAsUm64FBW7dKC2ECkpxfhKgesUJHXcSBlwiYifwxyIaillgrt2YVxVxV3soz5n2OPYlb0halqimLjLyKIobPhbRfGq4WxxXi7mH9hssmgxfN0dvbw32sf1IlfmothFSdMqt9OtsknbXSAn8XJg7gk/kVSU6W+MjIyMjP/KJxbFiwNr6IODAAAAAElFTkSuQmCC","orcid":"","institution":"University of Paris-Saclay","correspondingAuthor":true,"prefix":"","firstName":"Thibaud","middleName":"","lastName":"Dieudonné","suffix":""}],"badges":[],"createdAt":"2026-03-27 10:54:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9243799/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9243799/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105996183,"identity":"02a56f90-558d-4830-9473-ac6d348b8419","added_by":"auto","created_at":"2026-04-02 09:13:22","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":549932,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverall architecture of the ATP8B1-CDC50A flippase complex.\u003c/strong\u003e\u003cbr\u003e\n(\u003cstrong\u003eA\u003c/strong\u003e) Topological diagram of the flippase. Color code: the cytosolic A-, N-, and P-domains of ATP8B1 are colored yellow, red, and blue, respectively. The transmembrane domain of ATP8B1 is shown in wheat. The N- and C-terminal tails of ATP8B1 are colored light blue and green, respectively. CDC50A is colored pink. (\u003cstrong\u003eB\u003c/strong\u003e) Structure of the autoinhibited complex in the E2P\u003csub\u003eautoinhibited\u003c/sub\u003e\u0026nbsp;conformation (PDB: 7PY4), with the N- and C-terminal tails bound to the cytosolic domains of ATP8B1. Same color code as in panel A.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9243799/v1/44925518d88a7677170bf22c.jpeg"},{"id":105996147,"identity":"ad2892ed-1698-401d-bdbc-88c289dff704","added_by":"auto","created_at":"2026-04-02 09:13:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":682901,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe distal disordered region of the C-terminal tail of ATP8B1 contributes to the autoinhibition mechanism. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Detailed view of the different parts of the C-terminal tail of ATP8B1 interacting with the cytosolic domains of ATP8B1 in the E2P\u003csub\u003eautoinhibited\u003c/sub\u003e\u0026nbsp;conformation PDB: 7YP4. Color code as in Figure 1. For clarity purposes the N-terminal tail was omitted. (\u003cstrong\u003eB\u003c/strong\u003e) Schematic representation of the 1205-1251, 1215-1243 and 1215-1228 peptide sequences mimicking part of the C-terminal tail of ATP8B1. (\u003cstrong\u003eC\u003c/strong\u003e) Inhibition of ATP8B1 ATPase activity by increasing concentrations of the corresponding peptides. Data were fitted using a dose-response nonlinear regression curve, and the shaded area represents the 99% confidence interval of the fit. Error bars indicate the standard deviation of 3 technical replicates from 3 biological replicates (9 measurements in total). Refer to Table 1 for statistical analysis.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-9243799/v1/7c9fd4d172eae303a374e006.png"},{"id":105996199,"identity":"a45d1ea4-2f95-4e95-a37f-368baec006e1","added_by":"auto","created_at":"2026-04-02 09:13:26","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1084238,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe E243-R1228 salt bridge plays a critical role in the autoinhibition mechanism. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Close-up view of the interactions of the 1217-1228 region of the C-terminal tail of ATP8B1 with the A- and N- domains in the E2P autoinhibited conformation (PDB: 7YP4). Color code: the A-, N-domains of ATP8B1 are colored yellow and red, respectively. The C-terminal tail of ATP8B1 is colored in green. The salt bridge between E219 and R1228 is indicated by a dashed line. (\u003cstrong\u003eB\u003c/strong\u003e) Close-up view of the Van-der-Walls interactions between the A- and N- domain mediated by I1221 and I1227. A- and N- domain of ATP8B1 are shown as surface and colored by molecular lipophilicity potential (MLP) calculated in ChimeraX, with the fauchere method (Pettersen et al., 2021). (\u003cstrong\u003eC\u003c/strong\u003e) Schematic representation of the 1205-1251, 1205-1228, 1205-1227, and 1205-1226 peptide sequences mimicking part of the C-terminal tail of ATP8B1. (\u003cstrong\u003eD-E\u003c/strong\u003e) Inhibition of ATP8B1 ATPase activity by increasing concentrations of the corresponding peptides. Data were fitted using a dose-response nonlinear regression curve, and the shaded area represents the 99% confidence interval of the fit. Error bars indicate the standard deviation of 3 technical replicates from 3 biological replicates (9 measurements in total). Refer to Table 1 for statistical analysis.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-9243799/v1/492e375dd1679cc701622333.png"},{"id":105996155,"identity":"7f48c485-e8f2-4fae-bca5-5ed8bb0fcba2","added_by":"auto","created_at":"2026-04-02 09:13:15","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":215839,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe 1215-1228 fragment is sufficient to inhibit ATP8B1 ATPase activity. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Schematic representation of the 1205-1228, 1215-1228, and 1205-1215 peptide sequences mimicking part of the C-terminal tail of ATP8B1. (\u003cstrong\u003eB\u003c/strong\u003e) Inhibition of ATP8B1 ATPase activity by increasing concentrations of the corresponding peptides. Data were fitted using a dose-response nonlinear regression curve, and the shaded area represents the 99% confidence interval of the fit. Error bars indicate the standard deviation of 3 technical replicates from 3 biological replicates (9 measurements in total). Refer to Table 1 for statistical analysis.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-9243799/v1/00ec4b29705db71581ec7fa4.png"},{"id":107706683,"identity":"876c37dd-da51-4b48-9bfc-e615b2d25cac","added_by":"auto","created_at":"2026-04-24 09:18:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2629828,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9243799/v1/a24b136b-458b-488b-a2d2-142b6ef6a962.pdf"},{"id":105996145,"identity":"e0640e59-b55f-4e4b-b63b-99d23e8cea99","added_by":"auto","created_at":"2026-04-02 09:13:13","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":738927,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S1: Structural alignment of ATP8B1, ATP8A1 and Drs2 C-terminal tail regions. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Structural alignment of the most distal part of the C-terminal tails of ATP8B1 (green), ATP8A1 (light blue), and Drs2 (dark blue) as resolved in their respective cryoEM structures. ATP8B1 (PDB: 7PY4), ATP8A1 (PDB: 6K7L), and Drs2 (PDB: 6ROH) were aligned based on their N-domains. (\u003cstrong\u003eB\u003c/strong\u003e) Corresponding sequence alignment showing the Cα distances measured in ChimeraX. The two hydrophobic residues of the hydrophobic patch are underlined with a *.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-9243799/v1/841d252096fe9e65cad6e576.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mechanistic insights into autoinhibition of the human flippase ATP8B1","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMembranes of the late secretory pathway of eukaryotes are characterized by an asymmetric distribution of phospholipids. This asymmetry is defined, but not limited, by an enrichment of sphingolipids and phosphatidylcholine (PtdCho) in the exoplasmic leaflet, whereas phosphatidylserine (PtdSer), phosphatidylethanolamine (PtdEth) are mostly found in the cytosolic leaflet (Caputo et al., 2023). This asymmetry is essential for numerous cellular processes, in particular signaling and membrane trafficking events, where the enrichment of PtdSer on the cytoplasmic leaflet of the plasma membrane mediates the specific recruitment of peripheral proteins such as RAS small GTPases (Sakuragi \u0026amp; Nagata, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e, Leventis \u0026amp; Grinstein, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eP4-ATPases, also known as flippases, participate in the establishment and maintenance of this asymmetry by transporting specific phospholipids from the exoplasmic to the cytosolic leaflet of membranes, in an ATP-dependent manner (Sakuragi \u0026amp; Nagata, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). P4-ATPases are part of the P-type ATPase superfamily and therefore share a common architecture, with an α-helical transmembrane domain containing the lipid transport site, connected to three cytosolic domains: the nucleotide-binding (N) domain, which binds ATP; the phosphorylation (P) domain, which contains a conserved aspartate residue that is transiently phosphorylated during the transport cycle; and the actuator (A) domain, responsible for transporter dephosphorylation and return of the enzyme to its initial state (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) (Palmgren, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e, Stock et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In addition, most P4-ATPases form a binary complex with a subunit from the CDC50 family, which mediates their proper function and localization (Bryde et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The structural details of the lipid transport cycle of P4-type ATPases have been extensively studied by cryoEM and X-ray crystallography and follow the Post-Albers model, with two main phases: the E1 phase, in which the P4-ATPase undergoes phosphorylation in a lipid-independent manner, and the E2 phase, in which lipid binding and occlusion within the protein trigger dephosphorylation of the conserved catalytic aspartate in the P-domain by an invariant glutamate of the A-domain (Sai \u0026amp; Lee, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Duan \u0026amp; Li, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSeveral P4-ATPases have been shown to be tightly regulated by their N- and/or C-terminal extensions. In particular, the yeast flippase Drs2-Cdc50 and the human flippases ATP8A2-CDC50A and ATP8B1-CDC50A have been reported to be autoinhibited through their terminal extensions (Zhou et al, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Azouaoui et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Matsell et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Dieudonn\u0026eacute; et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). CryoEM structures of the Drs2-Cdc50 and ATP8B1-CDC50A complexes in both autoinhibited and active conformations revealed that the C-terminal tail of these P4-ATPases can establish extensive interactions with the three cytosolic domains of the enzyme after ATP hydrolysis, thereby locking the transporter in E2P autoinhibited conformations (Timcenko et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Timcenko et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Cheng et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Dieudonn\u0026eacute; et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Dieudonn\u0026eacute; et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In this state, the P4-ATPase is unable to occlude its substrate lipid, a step that is required for lipid translocation. In ATP8B1, this regulatory mechanism is further reinforced by the N-terminal tail, which inserts between the A and P domains once the C-terminal tail is engaged, further restricting the cytosolic domain rearrangements required to proceed through the catalytic lipid transport cycle (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) (Dieudonn\u0026eacute; et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Mutations or deletion of this C-terminal tail region in ATP8A2 or Drs2 have been shown to have a drastic effect on protein expression level, likely due to protein instability (Azouaoui et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, Matsell et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eInherited mutations in P4-ATPases have been associated with various human diseases (Shin and Takatsu, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Well-documented examples include progressive familial intrahepatic cholestasis (PFIC) type 1 and the less severe benign recurrent intrahepatic cholestasis (BRIC1), which both result directly from mutations in ATP8B1 (Klomp et al, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). While previous studies have investigated ATP8B1 lipid transport specificity and function (Muranaka et al, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Dieudonn\u0026eacute; et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), very few studies have explored the regulatory mechanisms of ATP8B1, in particular the role of the different regions of its C-terminal tail in the autoinhibition process.\u003c/p\u003e \u003cp\u003ePreviously, our group used purified ATP8B1-CDC50A lacking its C-terminal tail to perform trans-inhibition assays with synthetic peptides to investigate the role of the N- and C-terminal tails of ATP8B1, as well as the putative role of unknown kinase(s) in the activation mechanism of ATP8B1 by phosphorylation of S1223 (Dieudonn\u0026eacute; et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Here, we used a similar approach with peptides corresponding to fragments of the ATP8B1 C-terminal tail to gain further insight into the role of the different sections of this autoinhibitory region of the flippase. This approach allowed us to define the contribution of specific segments, providing a clearer description of the autoinhibition mechanism of ATP8B1 and, more broadly, of other flippases regulated in a similar manner.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePeptides\u003c/h2\u003e \u003cp\u003eAll Peptides presented is this study were synthetized by Biomatik Company (Canada) with \u0026gt;\u0026thinsp;90% purity grade, TFA free. The lyophilized peptides were directly resuspended in buffer A (50mM MOPS-Tris pH 7, 100 mM KCl) and stored at -20\u0026deg;C before use.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHeterologous co-expression and purification of the ATP8B1-CDC50A complex\u003c/h3\u003e\n\u003cp\u003eYeast cultures, recombinant protein expression and membrane preparation were performed as described previously (Azouaoui et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, Dieudonn\u0026eacute; et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Purification of C-terminal cleavable hATP8B1(HRV 3C protease L1185)-hCDC50A complex was performed as described previously (Dieudonn\u0026eacute; et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The final protein concentration of three independent biological triplicates was determined by loading duplicate samples from each purification on a Coomassie blue-stained SDS-PAGE, with purified Drs2-Cdc50 complex of known amount (determined by absorbance at 280 nm).\u003c/p\u003e\n\u003ch3\u003eATPase activity assay\u003c/h3\u003e\n\u003cp\u003eTo estimate the effect of treating C-terminally truncated ATP8B1 (Δ1185) - CDC50A complex with different peptides, ATP hydrolysis was measured using an enzyme-coupled assay (Sehgal et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). To measure the ATPase activity of C-terminally truncated ATP8B1 (Δ1185), samples were prepared containing 0.75 \u0026micro;g.mL⁻\u0026sup1; ATP8B1-CDC50A, 0.15 mg.mL⁻\u0026sup1; PtdEth, 0.025 mg.mL⁻\u0026sup1; PI(3,4,5)P\u003csub\u003e3\u003c/sub\u003e, 0.04 mg.mL⁻\u0026sup1; PK, 0.1 mg.mL⁻\u0026sup1; LDH, 1 mM PEP, 1 mg.mL⁻\u0026sup1; DDM, and 0.2 mg.mL⁻\u0026sup1; CHS in buffer A without glycerol. To allow proper lipid diffusion within flippase-containing detergent micelles, samples were incubated for 1 h 30 min at 4\u0026deg;C prior to the addition of 165 \u0026micro;M NADH. ATPase activity assays were performed in 96-well plates, with 350 \u0026micro;L sample per well. The decrease in absorbance at 340 nm was monitored for 8 min per run at 37\u0026deg;C using a SpectraMax\u0026reg; i3 microplate reader in kinetic mode. After an initial run for background measurement, ATP was added to a final concentration of 1 mM to measure ATPase activity prior to peptide addition. This was followed by up to three additional runs with increasing peptide concentrations.\u003c/p\u003e \u003cp\u003eTo assess peptide effects on ATPase activity, the activity measured in each run was normalized to the activity of the same sample before peptide addition, after background correction. Each peptide was tested at nine different concentrations ranging from 1 nM to 6.6 \u0026micro;M. Technical triplicates were performed for each concentration and for three independent biological replicates of purified C-terminally truncated ATP8B1 (Δ1185)-CDC50A, resulting in a total of nine measurements per peptide concentration.\u003c/p\u003e\n\u003ch3\u003eStatistics\u003c/h3\u003e\n\u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e of each peptide was estimated, when possible, from the 9 replicates of 9 different peptide concentrations. For each peptide, dose-response nonlinear regression curve fits, as well as the associated 99% confidence band, were generated using GraphPad Prism software based on the associated covariance matrix. For the non-linear fit, the top was constrained to the normalized activity of 1. The non-linear fits are shown in solid, and the 99% confidence bands are shown as transparent bands enclosed in dashed lines.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eThe distal disordered region of the C-terminal tail of ATP8B1 contributes to the autoinhibition mechanism\u003c/h2\u003e \u003cp\u003eTo gain insight into the structural basis of ATP8B1 autoinhibition mediated by its C-terminal region, we designed a series of peptides corresponding to different segments of the ATP8B1 C-terminal tail, named according to their position within ATP8B1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B). We then performed trans-inhibition assays of the ATPase activity of C-terminally truncated ATP8B1 (Δ1185) in detergent in the presence of the lipid substrate PtdEth and the activating lipid phosphatidylinositol trisphosphate (PI(3,4,5)P₃), and evaluated their inhibitory properties by determining IC\u003csub\u003e50\u003c/sub\u003e values (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). As previously observed, the peptide spanning residues 1205\u0026ndash;1251, corresponding to the full ATP8B1 C-terminal tail, efficiently inhibited ATP8B1 ATPase activity, with an IC\u003csub\u003e50\u003c/sub\u003e of 16 nM (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) (Dieudonn\u0026eacute; et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Truncation of the last eight residues (1205\u0026ndash;1243) moderately reduced inhibitory potency, yielding an IC\u003csub\u003e50\u003c/sub\u003e of 56 nM (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In contrast, removal of the region corresponding to the disordered segment of the C-terminal tail unresolved in ATP8B1 cryoEM structures (1205\u0026ndash;1228) resulted in an approximately tenfold increase in IC\u003csub\u003e50\u003c/sub\u003e to 175 nM, relative to full-length peptide 1205\u0026ndash;1251. These results indicate that the disordered portion of the ATP8B1 C-terminal tail contributes to autoinhibition, likely through transient interactions with the cytosolic domains.\u003c/p\u003e \u003cp\u003eNotably, ATP8A1 and ATP8A2 in contrast to ATP8B1 possess shorter C-terminal tails with smaller disordered distal segments. In the autoinhibited cryoEM structure of ATP8A1, only the last nine residues could not be modeled (Hiraizumi et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), compared with twenty-three residues in ATP8B1 (Dieudonn\u0026eacute; et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Compared to ATP8B1 and the yeast homolog Drs2, ATP8A1 and ATP8A2 do show ATPase activity for the full-length proteins (Coleman et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Hiraizumi et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Matsell et al., 2024; Azouaoui et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Dieudonn\u0026eacute; et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eThe E243-R1228 interaction is critical for ATP8B1 autoinhibition\u003c/h3\u003e\n\u003cp\u003eThe most distal region of the ATP8B1 C-terminus observed in the cryoEM structures interacts directly with the A-domain through a salt bridge (E219-R1228) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) and with the A- and N-domain through Van-der-Walls interactions in a hydrophobic patch formed by Y225, F239, M538, I593, I1225 and I1227 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Hence, we evaluated the inhibitory properties of shorter peptides lacking either R1228 (1205\u0026ndash;1227) or R1228 and I1227 (1205\u0026ndash;1226) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Removal of R1228 resulted in a fivefold increase in IC\u003csub\u003e50\u003c/sub\u003e, from 175 nM for peptide 1205\u0026ndash;1228 to 907 nM for peptide 1205\u0026ndash;1227 while the additional removal of I1227 results in an almost complete loss of the inhibition properties of the corresponding 1205\u0026ndash;1226 peptide (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo further assess the contribution of the E219-R1228 interaction, we tested the full-length C-terminal peptide in which R1228 was substituted with alanine (1205\u0026ndash;1251 R1228A). This mutant peptide exhibited a 19-fold increase in IC\u003csub\u003e50\u003c/sub\u003e (305 nM) compared to the WT 1205\u0026ndash;1251 peptide, indicating a major contribution of this residue to the autoinhibition (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). However, despite the reduced potency of the R1228A mutant peptide, it retained a stronger inhibitory activity than peptide 1205\u0026ndash;1227, with an IC\u003csub\u003e50\u003c/sub\u003e of 307 nM versus 907 nM, respectively, suggesting that residues located C-terminally to R1228 may partially compensate for the loss of this interaction. In particular, K1229, K1230, and R1231 could potentially engage in electrostatic interactions with E219, consistent with the apparent flexibility of this region of the C-terminal tail.\u003c/p\u003e \u003cp\u003eInterestingly, structural alignment of the C-terminal regions of ATP8B1, ATP8A1, and Drs2 shows that R1228 in ATP8B1 is not conserved (Figure S1). Instead, in ATP8A1 and Drs2, which exhibit a higher degree of sequence and structural conservation with each other, the interaction with the conserved glutamate in the A-domain (E219 in ATP8B1) appears to be mediated by a hydrogen bond involving a conserved tyrosine residue (Y1292 and Y1139 for Drs2 and ATP8A1, respectively). Furthermore, the 1205\u0026ndash;1251 peptide corresponding to the ATP8B1 C-terminal tail does not inhibit Drs2 ATPase activity (Dieudonn\u0026eacute; et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In contrast to R1228, E219 is highly conserved among P4-ATPases, and not only in those displaying a long C-terminal tail harbouring a (G/A)(Y/F)AFS motif. In E1 states, it mediates intra-A-domain interactions of ATP8A1, Drs2 and even ATP11C.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eThe 1216–1228 C-terminal region bridging the A- and N- domains is sufficient for autoinhibition\u003c/h3\u003e\n\u003cp\u003eFinally, we wanted to investigate the role of the highly conserved (G/A)(Y/F)AFS motif, known to interact with the ATP binding site of the N-domain in the E2P\u003csub\u003eautoinhibited\u003c/sub\u003e conformation. To this end, we designed two peptides: one spanning residues 1205\u0026ndash;1215, containing the conserved motif along with additional polar and positively charged residues to ensure peptide solubility, and a second peptide corresponding to residues 1216\u0026ndash;1228, corresponding to the region of the C-terminal tail of ATP8B1 intercalated in between the N- and A-domains (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eThe 1205\u0026ndash;1215 peptide did not inhibit ATPase activity, whereas peptide 1216\u0026ndash;1228 retained inhibitory properties with an IC\u003csub\u003e50\u003c/sub\u003e of 445 nM. Although the isolated (G/A)(Y/F)AFS conserved motif (1205\u0026ndash;1215) was not sufficient to directly inhibit ATPase activity, its presence markedly enhanced inhibition in the context of the longer peptide (1205\u0026ndash;1228) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Consistently, peptide 1205\u0026ndash;1228 exhibited a substantially lower IC\u003csub\u003e50\u003c/sub\u003e of 147 nM, approximately three-fold lower than that of peptide 1216\u0026ndash;1228. Together, these results indicate that the conserved (G/A)(Y/F)AFS motif is not sufficient for inhibition on its own but enhances the inhibitory capacity of the adjacent C-terminal region that intercalates the N- and A-domains.\u003c/p\u003e \u003cp\u003eThese results are also in line with previous work done on the yeast flippase Drs2 where truncation of its C-terminal tail by limited proteolysis was used to relieve the autoinhibition mechanism \u003cem\u003ein vitro\u003c/em\u003e (Azouaoui et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Indeed, the truncation by limited proteolysis of Drs2 leaves the (G/A)(Y/F)AFS motif while removing the last 64 residues of the Drs2 C-terminal tail, and results in an active flippase consuming ATP in a lipid-dependent manner.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eFrom a structural perspective, it has been shown that autoinhibited ATP8B1 is locked in an E2P-like conformation, with a possible equilibrium between states with an open or closed lipid-binding site (Dieudonn\u0026eacute; et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Based on this observation, we and others have proposed that C-terminal mediated autoinhibition restricts the rotation of the A-domain, a movement that normally results from structural rearrangements of TM1 and TM2 induced by lipid head-group binding prior to its occlusion within the transport site. Here, our data shows that the minimal region of the ATP8B1 C-terminal tail responsible for autoinhibition corresponds to the segment that most strongly interacts with the A domain, in line with that model.\u003c/p\u003e \u003cp\u003eMore importantly, the A-domain rotation that is blocked by the inhibitory peptide represents a conformational transition, which is highly conserved among P-type ATPases and directly linked to the dephosphorylation of the catalytic aspartate in the P-domain during the transport cycle. Given the strong conservation of this mechanistic step across the P-type ATPase superfamily, our results may have broader implications beyond ATP8B1. In particular, the identification of a minimal peptide segment capable of restricting A-domain motion suggests a potential strategy for the design of inhibitory peptides targeting other P-type ATPases. Conversely, a detailed understanding of this autoinhibitory interaction could also guide the rational design of molecules aimed at antagonizing C-terminal-mediated autoinhibition in P4-ATPases, thereby promoting enzyme activation. Finally, although our results provide important insights into the autoinhibition mechanism of ATP8B1 and P4-ATPases more broadly, how this autoinhibition is relieved \u003cem\u003ein vivo\u003c/em\u003e remains to be determined.\u003c/p\u003e \u003cp\u003eTable 1 Statistical analysis of the inhibition properties of the peptides mimicking different regions of the C-terminal tail of ATP8B1. ND: Not determined.\u003c/p\u003e \u003cp\u003e\u003cimg 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\" width=\"724\" height=\"306\"\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting Interests\u003c/h2\u003e \u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eacquisition - PN TD\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization - PN TDMethodology - CS TDInvestigation - MJL MRFormal analysis - MJL CS TDWriting original draft - MJLWriting review and editing - MR PN CS TDVisualization - MJL MR CS TDSupervision / project administration - PN CS TDFunding acquisition - PN TD\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank Joseph Lyons, Amelie Benfeldt Purup, Filip Pamula, Line Marie Christiansen, Guillaume Lenoir, C\u0026eacute;dric Montigny, and Poul S\u0026oslash;rensen for fruitful discussions and insightful feedback throughout this project. We are also grateful to Anna Marie Nielsen and Karen Bech-Pedersen for their excellent technical and administrative assistance.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data used to support the findings of this study are available from the corresponding authors upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAzouaoui H, Montigny C, Dieudonn\u0026eacute; T, Champeil P, Jacquot A, V\u0026aacute;zquez-Ibar JL, Mar\u0026eacute;chal L, Ulstrup P, Ash J, Lyons M-R, Nissen JA, P., Lenoir G (2017) High phosphatidylinositol 4-phosphate (PI4P)-dependent ATPase activity for the Drs2p-Cdc50p flippase after removal of its N- and C-terminal extensions. 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J Biol Chem 288(44):31807\u0026ndash;31815. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1074/jbc.M113.481986\u003c/span\u003e\u003cspan address=\"10.1074/jbc.M113.481986\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"the-journal-of-membrane-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jmbi","sideBox":"Learn more about [The Journal of Membrane Biology](http://link.springer.com/journal/232)","snPcode":"232","submissionUrl":"https://submission.nature.com/new-submission/232/3","title":"The Journal of Membrane Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"P-type ATPase, Flippases, P4-ATPase, Autoinhibition, ATP8B1, PFIC1, Intrahepatic cholestasis","lastPublishedDoi":"10.21203/rs.3.rs-9243799/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9243799/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eP4-ATPases are lipid flippases that maintain membrane phospholipid asymmetry by transporting specific phospholipids from the exoplasmic to the cytosolic leaflet, an essential process for membrane integrity, trafficking and signaling. Several P4-ATPases are tightly regulated by autoinhibitory N- and C-terminal extensions, yet the molecular basis of this regulation remains incompletely understood. Here, we investigated the autoinhibition mechanism of the human flippase ATP8B1 using trans-inhibition assays with synthetic peptides derived from its C-terminal tail. Using purified C-terminally truncated ATP8B1-CDC50A, we systematically assessed the inhibitory properties of peptides corresponding to distinct segments of the C-terminus. We show that the distal disordered region of the C-terminal tail significantly contributes to autoinhibition, likely through transient interactions with the cytosolic domains. We further identify a critical interaction between R1228 in the C-terminal tail and E219 in the A-domain, whose disruption markedly reduces inhibitory potency. In addition, we demonstrate that a minimal peptide spanning residues 1216\u0026ndash;1228, which bridges the A- and N-domains in the autoinhibited conformation, is sufficient to inhibit ATPase activity. Together, these results refine the molecular description of ATP8B1 autoinhibition, open the way for structure-based activation strategies and provide insight into conserved regulatory mechanisms among P4-ATPases.\u003c/p\u003e","manuscriptTitle":"Mechanistic insights into autoinhibition of the human flippase ATP8B1","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-02 09:10:47","doi":"10.21203/rs.3.rs-9243799/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-28T23:26:25+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-28T21:31:02+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-27T09:04:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"293857824147277945176804596082058269876","date":"2026-04-01T13:58:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"26628314153905183133580431408980595552","date":"2026-03-31T18:21:23+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-29T10:25:23+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-28T01:40:45+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-28T01:40:25+00:00","index":"","fulltext":""},{"type":"submitted","content":"The Journal of Membrane Biology","date":"2026-03-27T10:38:22+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"the-journal-of-membrane-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jmbi","sideBox":"Learn more about [The Journal of Membrane Biology](http://link.springer.com/journal/232)","snPcode":"232","submissionUrl":"https://submission.nature.com/new-submission/232/3","title":"The Journal of Membrane Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"945a7307-175f-45f0-9507-261cbee528b9","owner":[],"postedDate":"April 2nd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-04-28T23:38:45+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-02 09:10:47","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9243799","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9243799","identity":"rs-9243799","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}