Conformational flexibility of apolipoprotein A-I amino- and carboxy-termini is necessary for lipid binding but not cholesterol efflux
Because of its critical role in HDL formation, significant efforts have been devoted to studying apolipoprotein A-I (APOA1) structural transitions in response to lipid binding. To assess the requirements for the conformational freedom of its termini during HDL particle formation, we generated three...
| Main Authors: | , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2022-03-01
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| Series: | Journal of Lipid Research |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S0022227522000013 |
| _version_ | 1818772806372950016 |
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| author | Shimpi Bedi Jamie Morris Amy Shah Rachel C. Hart W. Gray Jerome Stephen G. Aller Chongren Tang Tomas Vaisar Karin E. Bornfeldt Jere P. Segrest Jay W. Heinecke W. Sean Davidson |
| author_facet | Shimpi Bedi Jamie Morris Amy Shah Rachel C. Hart W. Gray Jerome Stephen G. Aller Chongren Tang Tomas Vaisar Karin E. Bornfeldt Jere P. Segrest Jay W. Heinecke W. Sean Davidson |
| author_sort | Shimpi Bedi |
| collection | DOAJ |
| description | Because of its critical role in HDL formation, significant efforts have been devoted to studying apolipoprotein A-I (APOA1) structural transitions in response to lipid binding. To assess the requirements for the conformational freedom of its termini during HDL particle formation, we generated three dimeric APOA1 molecules with their termini covalently joined in different combinations. The dimeric (d)-APOA1C-N mutant coupled the C-terminus of one APOA1 molecule to the N-terminus of a second with a short alanine linker, whereas the d-APOA1C-C and d-APOA1N-N mutants coupled the C-termini and the N-termini of two APOA1 molecules, respectively, using introduced cysteine residues to form disulfide linkages. We then tested the ability of these constructs to generate reconstituted HDL by detergent-assisted and spontaneous phospholipid microsolubilization methods. Using cholate dialysis, we demonstrate WT and all APOA1 mutants generated reconstituted HDL particles of similar sizes, morphologies, compositions, and abilities to activate lecithin:cholesterol acyltransferase. Unlike WT, however, the mutants were incapable of spontaneously solubilizing short chain phospholipids into discoidal particles. We found lipid-free d-APOA1C-N and d-APOA1N-N retained most of WT APOA1’s ability to promote cholesterol efflux via the ATP binding cassette transporter A1, whereas d-APOA1C-C exhibited impaired cholesterol efflux. Our data support the double belt model for a lipid-bound APOA1 structure in nascent HDL particles and refute other postulated arrangements like the “double super helix.” Furthermore, we conclude the conformational freedom of both the N- and C-termini of APOA1 is important in spontaneous microsolubilization of bulk phospholipid but is not critical for ABCA1-mediated cholesterol efflux. |
| first_indexed | 2024-12-18T10:15:12Z |
| format | Article |
| id | doaj.art-85e2394e2ac54dddbe32143770942ea7 |
| institution | Directory Open Access Journal |
| issn | 0022-2275 |
| language | English |
| last_indexed | 2024-12-18T10:15:12Z |
| publishDate | 2022-03-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Journal of Lipid Research |
| spelling | doaj.art-85e2394e2ac54dddbe32143770942ea72022-12-21T21:11:20ZengElsevierJournal of Lipid Research0022-22752022-03-01633100168Conformational flexibility of apolipoprotein A-I amino- and carboxy-termini is necessary for lipid binding but not cholesterol effluxShimpi Bedi0Jamie Morris1Amy Shah2Rachel C. Hart3W. Gray Jerome4Stephen G. Aller5Chongren Tang6Tomas Vaisar7Karin E. Bornfeldt8Jere P. Segrest9Jay W. Heinecke10W. Sean Davidson11Department of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, OH, USADepartment of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, OH, USADepartment of Pediatrics, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USADepartment of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN, USADepartment of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN, USADepartment of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, AL, USADepartment of Medicine, University of Washington School of Medicine, Seattle, WA, USADepartment of Medicine, University of Washington School of Medicine, Seattle, WA, USADepartment of Medicine, University of Washington School of Medicine, Seattle, WA, USADepartment of Medicine, Vanderbilt University School of Medicine, Nashville, TN, USADepartment of Medicine, University of Washington School of Medicine, Seattle, WA, USADepartment of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, OH, USA; For correspondence: W. Sean DavidsonBecause of its critical role in HDL formation, significant efforts have been devoted to studying apolipoprotein A-I (APOA1) structural transitions in response to lipid binding. To assess the requirements for the conformational freedom of its termini during HDL particle formation, we generated three dimeric APOA1 molecules with their termini covalently joined in different combinations. The dimeric (d)-APOA1C-N mutant coupled the C-terminus of one APOA1 molecule to the N-terminus of a second with a short alanine linker, whereas the d-APOA1C-C and d-APOA1N-N mutants coupled the C-termini and the N-termini of two APOA1 molecules, respectively, using introduced cysteine residues to form disulfide linkages. We then tested the ability of these constructs to generate reconstituted HDL by detergent-assisted and spontaneous phospholipid microsolubilization methods. Using cholate dialysis, we demonstrate WT and all APOA1 mutants generated reconstituted HDL particles of similar sizes, morphologies, compositions, and abilities to activate lecithin:cholesterol acyltransferase. Unlike WT, however, the mutants were incapable of spontaneously solubilizing short chain phospholipids into discoidal particles. We found lipid-free d-APOA1C-N and d-APOA1N-N retained most of WT APOA1’s ability to promote cholesterol efflux via the ATP binding cassette transporter A1, whereas d-APOA1C-C exhibited impaired cholesterol efflux. Our data support the double belt model for a lipid-bound APOA1 structure in nascent HDL particles and refute other postulated arrangements like the “double super helix.” Furthermore, we conclude the conformational freedom of both the N- and C-termini of APOA1 is important in spontaneous microsolubilization of bulk phospholipid but is not critical for ABCA1-mediated cholesterol efflux.http://www.sciencedirect.com/science/article/pii/S0022227522000013apolipoprotein A-IATP binding cassette transporter ABCA1cholesterol effluxelectron microscopyhigh-density lipoproteinlecithin:cholesterol acyltransferase |
| spellingShingle | Shimpi Bedi Jamie Morris Amy Shah Rachel C. Hart W. Gray Jerome Stephen G. Aller Chongren Tang Tomas Vaisar Karin E. Bornfeldt Jere P. Segrest Jay W. Heinecke W. Sean Davidson Conformational flexibility of apolipoprotein A-I amino- and carboxy-termini is necessary for lipid binding but not cholesterol efflux Journal of Lipid Research apolipoprotein A-I ATP binding cassette transporter ABCA1 cholesterol efflux electron microscopy high-density lipoprotein lecithin:cholesterol acyltransferase |
| title | Conformational flexibility of apolipoprotein A-I amino- and carboxy-termini is necessary for lipid binding but not cholesterol efflux |
| title_full | Conformational flexibility of apolipoprotein A-I amino- and carboxy-termini is necessary for lipid binding but not cholesterol efflux |
| title_fullStr | Conformational flexibility of apolipoprotein A-I amino- and carboxy-termini is necessary for lipid binding but not cholesterol efflux |
| title_full_unstemmed | Conformational flexibility of apolipoprotein A-I amino- and carboxy-termini is necessary for lipid binding but not cholesterol efflux |
| title_short | Conformational flexibility of apolipoprotein A-I amino- and carboxy-termini is necessary for lipid binding but not cholesterol efflux |
| title_sort | conformational flexibility of apolipoprotein a i amino and carboxy termini is necessary for lipid binding but not cholesterol efflux |
| topic | apolipoprotein A-I ATP binding cassette transporter ABCA1 cholesterol efflux electron microscopy high-density lipoprotein lecithin:cholesterol acyltransferase |
| url | http://www.sciencedirect.com/science/article/pii/S0022227522000013 |
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