First eight residues of apolipoprotein A-I mediate the C-terminus control of helical bundle unfolding and its lipidation

Autoři: Gregory Brubaker aff001;  Shuhui W. Lorkowski aff001;  Kailash Gulshan aff001;  Stanley L. Hazen aff001;  Valentin Gogonea aff001;  Jonathan D. Smith aff001
Působiště autorů: Department of Cardiovascular and Metabolic Sciences, Cleveland Clinic, Cleveland, Ohio, United States of America aff001;  Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, Ohio, United States of America aff002;  Department of Chemistry, Cleveland State University, Cleveland, Ohio, United States of America aff003
Vyšlo v časopise: PLoS ONE 15(1)
Kategorie: Research Article


The crystal structure of a C-terminal deletion of apolipoprotein A-I (apoA1) shows a large helical bundle structure in the amino half of the protein, from residues 8 to 115. Using site directed mutagenesis, guanidine or thermal denaturation, cell free liposome clearance, and cellular ABCA1-mediated cholesterol efflux assays, we demonstrate that apoA1 lipidation can occur when the thermodynamic barrier to this bundle unfolding is lowered. The absence of the C-terminus renders the bundle harder to unfold resulting in loss of apoA1 lipidation that can be reversed by point mutations, such as Trp8Ala, and by truncations as short as 8 residues in the amino terminus, both of which facilitate helical bundle unfolding. Locking the bundle via a disulfide bond leads to loss of apoA1 lipidation. We propose a model in which the C-terminus acts on the N-terminus to destabilize this helical bundle. Upon lipid binding to the C-terminus, Trp8 is displaced from its interaction with Phe57, Arg61, Leu64, Val67, Phe71, and Trp72 to destabilize the bundle. However, when the C-terminus is deleted, Trp8 cannot be displaced, the bundle cannot unfold, and apoA1 cannot be lipidated.

Klíčová slova:

Crystal structure – Disulfide bonds – Cholesterol – Lipid structure – Lipids – Liposomes – Point mutation – Guanidines


1. Wang S, Smith JD. ABCA1 and nascent HDL biogenesis. BioFactors. 2014;40: 547–554. doi: 10.1002/biof.1187 25359426

2. Jonas A, Drengler SM. Kinetics and Mechanism of Apolipoprotein A-I Interaction with Dimyristoylphosphatidylcholine Vesicles. J Biol Chem. 1980;255: 2190–2194. 7354087

3. Pownall HJ, Massey JB, Kusserow SK, Gotto AM. Kinetics of Lipid-Protein Interactions: Interaction of Apolipoprotein A-I from Human Plasma High Density Lipoproteins with Phosphatidylcholines. Biochemistry. 1978;17: 1183–1188. doi: 10.1021/bi00600a008 207309

4. Sorci-Thomas M, Kearns MW, Lee JP. Apolipoprotein A-I domains involved in lecithin-cholesterol acyltransferase activation. Structure:function relationships. J Biol Chem. 1993;268: 21403–21409. 8407982

5. Remaley AT, Thomas F, Stonik JA, Demosky SJ, Bark SE, Neufeld EB, et al. Synthetic amphipathic helical peptides promote lipid efflux from cells by an ABCA1-dependent and an ABCA1-independent pathway. J Lipid Res. 2003;44: 828–836. doi: 10.1194/jlr.M200475-JLR200 12562845

6. Mei X, Atkinson D. Crystal structure of C-terminal truncated apolipoprotein A-I reveals the assembly of high density lipoprotein (HDL) by dimerization. J Biol Chem. 2011;286: 38570–82. doi: 10.1074/jbc.M111.260422 21914797

7. Melchior JT, Walker RG, Cooke AL, Morris J, Castleberry M, Thompson TB, et al. A consensus model of human apolipoprotein A-I in its monomeric and lipid-free state. Nat Struct Mol Biol. 2017;24: 1093–1099. doi: 10.1038/nsmb.3501 29131142

8. Ji Y, Jonas A. Properties of an N-terminal proteolytic fragment of apolipoprotein AI in solution and in reconstituted high density lipoproteins. J Biol Chem. 1995;270: 11290–11297. doi: 10.1074/jbc.270.19.11290 7744765

9. Davidson WS, Hazlett T, Mantulin WW, Jonas a. The role of apolipoprotein AI domains in lipid binding. Proc Natl Acad Sci U S A. 1996;93: 13605–13610. doi: 10.1073/pnas.93.24.13605 8942981

10. Chroni A, Liu T, Gorshkova I, Kan H-Y, Uehara Y, von Eckardstein A, et al. The Central Helices of ApoA-I Can Promote ATP-binding Cassette Transporter A1 (ABCA1)-mediated Lipid Efflux. J Biol Chem. 2003;278: 6719–6730. doi: 10.1074/jbc.M205232200 12488454

11. Burgess JW, Frank PG, Franklin V, Liang P, McManus DC, Desforges M, et al. Deletion of the C-terminal domain of apolipoprotein A-I impairs cell surface binding and lipid efflux in macrophage. Biochemistry. 1999;38: 14524–14533. doi: 10.1021/bi990930z 10545174

12. Beckstead JA, Block BL, Bielicki JK, Kay CM, Oda MN, Ryan RO. Combined N- and C-terminal truncation of human apolipoprotein A-I yields a folded, functional central domain. Biochemistry. 2005;44: 4591–4599. doi: 10.1021/bi0477135 15766290

13. Koyama M, Tanaka M, Dhanasekaran P, Lund-Katz S, Phillips MC, Saito H. Interaction between the N- and C-terminal domains modulates the stability and lipid binding of apolipoprotein A-I. Biochemistry. 2009;48: 2529–37. doi: 10.1021/bi802317v 19239199

14. Saito H, Dhanasekaran P, Nguyen D, Holvoet P, Lund-Katz S, Phillips MC. Domain structure and lipid interaction in human apolipoproteins A-I and E, a general model. J Biol Chem. 2003;278: 23227–23232. doi: 10.1074/jbc.M303365200 12709430

15. Mei X, Liu M, Herscovitz H, Atkinson D. Probing the C-terminal domain of lipid-free apoA-I demonstrates the vital role of the H10B sequence repeat in HDL formation. J Lipid Res. 2016;57: 1507–1517. doi: 10.1194/jlr.M068874 27317763

16. Gross E, Peng DQ, Hazen SL, Smith JD. A novel folding intermediate state for apolipoprotein A-I: Role of the amino and carboxy termini. Biophys J. 2006;90: 1362–1370. doi: 10.1529/biophysj.105.075069 16326917

17. Ryan RO, Forte TM, Oda MN. Optimized bacterial expression of human apolipoprotein A-I. Protein Expr Purif. 2003;27: 98–103. doi: 10.1016/s1046-5928(02)00568-5 12509990

18. Sparks DL, Lund-Katz S, Phillips MC. The charge and structural stability of apolipoprotein A-I in discoidal and spherical recombinant high density lipoprotein particles. J Biol Chem. 1992;267: 25839–25847. 1464598

19. Saito H, Dhanasekaran P, Nguyen D, Deridder E, Holvoet P, Lund-Katz S, et al. α-helix formation is required for high affinity binding of human apolipoprotein A-I to lipids. J Biol Chem. 2004;279: 20974–20981. doi: 10.1074/jbc.M402043200 15020600

20. Tanaka M, Koyama M, Dhanasekaran P, Nguyen D, Nickel M, Lund-Katz S, et al. Influence of tertiary structure domain properties on the functionality of apolipoprotein A-I. Biochemistry. 2008;47: 2172–2180. doi: 10.1021/bi702332b 18205410

21. Tanaka M, Dhanasekaran P, Nguyen D, Nickel M, Takechi Y, Lund-Katz S, et al. Influence of N-terminal helix bundle stability on the lipid-binding properties of human apolipoprotein A-I. Biochim Biophys Acta. 2011;1811: 25–30. doi: 10.1016/j.bbalip.2010.10.003 21040803

22. Silva RAGD, Hilliard GM, Fang J, Macha S, Davidson WS. A three-dimensional molecular model of lipid-free apolipoprotein A-I determined by cross-linking/mass spectrometry and sequence threading. Biochemistry. 2005;44: 2759–2769. doi: 10.1021/bi047717+ 15723520

23. Pollard RD, Fulp B, Samuel MP, Sorci-Thomas MG, Thomas MJ. The conformation of lipid-free human apolipoprotein A-I in solution. Biochemistry. 2013;52: 9470–81. doi: 10.1021/bi401080k 24308268

24. Segrest JP, Jones MK, Shao B, Heinecke JW. An Experimentally Robust Model of Monomeric Apolipoprotein A—I Created from a Chimera of Two X—ray Structures and Molecular Dynamics Simulations. Biochemistry. 2014;53: 7625–7640. doi: 10.1021/bi501111j 25423138

25. Oda MN, Forte TM, Ryan RO, Voss JC. The C-terminal domain of apolipoprotein A-I contains a lipid-sensitive conformational trigger. Nat Struct Biol. 2003;10: 455–460. doi: 10.1038/nsb931 12754494

26. Liu M, Mei X, Herscovitz H, Atkinson D. N-terminal mutation of apoA-I and interaction with ABCA1 reveal mechanisms of nascent HDL biogenesis. J Lipid Res. 2019;60: 44–75. doi: 10.1194/jlr.M084376 30249788

27. Peng DQ, Brubaker G, Wu Z, Zheng L, Willard B, Kinter M, et al. Apolipoprotein A-I tryptophan substitution leads to resistance to myeloperoxidase-mediated loss of function. Arterioscler Thromb Vasc Biol. 2008;28: 2063–2070. doi: 10.1161/ATVBAHA.108.173815 18688016

28. Huang Y, Didonato JA, Levison BS, Schmitt D, Li L, Wu Y, et al. An abundant dysfunctional apolipoprotein A1 in human atheroma. Nat Med. 2014;20: 193–203. doi: 10.1038/nm.3459 24464187

29. Gulshan K, Brubaker G, Conger H, Wang S, Zhang R, Hazen SL, et al. PI(4,5)P2 is Translocated by ABCA1 to the Cell Surface Where It Mediates Apolipoprotein A1 Binding and Nascent HDL Assembly. Circ Res. 2016;119: 827–838. doi: 10.1161/CIRCRESAHA.116.308856 27514935

30. Wang S, Gulshan K, Brubaker G, Hazen SL, Smith JD. ABCA1 mediates unfolding of apolipoprotein ai n terminus on the cell surface before lipidation and release of nascent high-density lipoprotein. Arterioscler Thromb Vasc Biol. 2013;33: 1197–1205. doi: 10.1161/ATVBAHA.112.301195 23559627

Článek vyšel v časopise


2020 Číslo 1
Nejčtenější tento týden