An N-Terminal Three-Helix Fragment of the Exchangeable Insect Apolipoprotein Apolipophorin III Conserves the Lipid Binding Properties of Wild-Type Protein

Dettloff, Matthias; Weers, Paul M.M.; Niere, Marc; Kay, Cyril M.; Ryan, Robert O.; Wiesner, Andreas

doi:10.1021/bi0013804

Cited by 19 publications

(31 citation statements)

References 41 publications

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“…Similar results were reported for the two-helix truncation variants of G. mellonella apoLp-III. 30-31 The increase in phospholipid binding activity is in good agreement with previous studies where it was shown that helix bundle stability inversely correlates with phospholipid vesicle solubilization. 36-40 Conceivably, the equilibrium between open and closed helix bundle states may have shifted to a more open conformation, leading to increased exposure of hydrophobic regions of the amphipathic α-helices, thereby promoting lipid binding.…”

Section: Discussionsupporting

confidence: 91%

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Deletion of the N- or C-Terminal Helix of Apolipophorin III To Create a Four-Helix Bundle Protein

et al. 2016

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Apolipophorin III (apoLp-III) is an exchangeable apolipoprotein found in insects and plays an important function in lipid transport. The protein has an unusual five-helix bundle architecture, deviating from the common four-helix bundle motif. To understand the role of the additional helix in apoLp-III, the N-terminal or C-terminal helix was deleted to create a putative four-helix bundle protein. While the protein lacking helix-1 could be expressed in bacteria albeit at reduced yields, apoLp-III lacking helix-5 could not be produced. Mutational analysis by truncating helix-5 showed that a minimum segment of approximately one third of the C-terminal helix is required for protein expression. The variant lacking helix-5 was produced by inserting a methionine residue between helix-4 and -5; subsequent cyanogenbromide cleavage generated the four-helix variant. Both N- and C-terminal helix deletion variants displayed significantly reduced helical content, protein stability, and tertiary structure. Despite the significantly altered structure, the variants were still fully functional. The rate of dimyristoylphosphatidylcholine vesicle solubilization was enhanced 4- to 5-fold compared to the wild-type protein, and the deletion variants were effective in binding to lipolyzed low density lipoprotein thereby preventing lipoprotein aggregation. These results show that the additional helix of apoLp-III is not essential for lipid binding, but is required for proper folding to keep the protein into a stable conformation.

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Section: Discussionsupporting

confidence: 91%

“…30-31 These protein constructs displayed a high degree of dimers and trimers of this otherwise monomeric protein. Therefore, self-association of the L. migratoria apoLp-III variants was assessed using DMS cross-linker.…”

Section: Resultsmentioning

confidence: 99%

Deletion of the N- or C-Terminal Helix of Apolipophorin III To Create a Four-Helix Bundle Protein

et al. 2016

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“…One of the studies concluded that the lipoproteins contain six molecules of apoLp-III (27), whereas the second report indicated the presence of five apolipoprotein molecules per discoidal particle (28). Our model would not be fully correct if the discoidal lipoproteins contain five molecules of apoLp-III.…”

Section: Spatial Arrangement Of Different Apolipoproteins Molecules Imentioning

confidence: 80%

“…These results indicate that the distances of separation between the helix 4 and the helices 1, 2, 3, and 5 increase significantly in the lipid-bound state. However, due to the small size of the discoidal lipoproteins and the presence of five or six molecules of apolipoprotein per lipoprotein particle (27)(28), a potential intermolecular energy transfer between Trp and probes located in different apoLp-III molecules was considered and investigated. This type of energy transfer, which could occur between donor (Trp) and acceptors located in different apolipoprotein molecules, could potentially lead to an underestimation of the distances of separation between donor and acceptor.…”

Section: Efficiency Of Fret and D-a Distances From Trp-113 (Helix 4) mentioning

confidence: 99%

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Structure of Apolipophorin-III in Discoidal Lipoproteins

Garda

Arrese

Soulages

2002

Journal of Biological Chemistry

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The structure of apolipophorin III in the lipid-bound state and the extent of the conformational change that takes place when the five-helix bundle apolipoprotein binds to a lipoprotein lipid surface were investigated by fluorescence resonance energy transfer in discoidal lipoproteins. Four intramolecular interhelical distances between helix pairs 1-4, 2-4, 3-4, and 5-4 were estimated by fluorescence resonance energy transfer in both the lipid-free and the lipid-bound states. Depending on the helices pairs, the intramolecular interhelical distances increased between 15 and > 20 Å upon binding of the apolipoprotein to lipid, demonstrating for the first time that binding to lipid is accompanied by a major change in interhelical distances. Using discoidal lipoproteins made with a combination of apolipophorin III molecules containing donor and acceptor groups and apolipophorin III molecules containing neither donor nor acceptor groups, it was possible to obtain information about intermolecular interhelical distances between the helix 4 of one apolipoprotein and the helices 1, 2, 3, and 5 of a second apolipoprotein residing in the same discoidal lipoprotein. Altogether, the estimated intermolecular and intramolecular interhelical distances suggest a model in which the apolipoprotein arranges in pairs of antiparallel and fully extended polypeptide chains surrounding the periphery of the bilayer disc.Exchangeable apolipoproteins represent a group of proteins characterized by their ability to reversibly bind to lipoprotein lipid surfaces (1). These apolipoproteins play a key role in the regulation of lipid metabolism (2). The interaction of lipoproteins with receptors (3-4) and the exchange of lipids between lipoproteins and lipoproteins and cells (5-7) are among the processes regulated by the structure of apolipoproteins in both the lipid-free and the lipid-bound states. A full understanding of the function of exchangeable apolipoproteins in the homeostasis of lipid metabolism and associated pathologies requires determining the structure of the protein in the lipid-bound state.Apolipophorin-III is a 17-kDa exchangeable apolipoprotein that is involved in the transport of lipids in the hemolymph of insects (8 -9). The structure of apoLp-III 1 in the lipid-free state was solved by x-ray crystallography in 1991 (10). ApoLp-III is composed of a bundle of five amphipathic ␣-helices connected by short loops (10). The study of the structure-function relationship of Locusta migratoria apoLp-III is of interest because the knowledge of the crystal structure of the full-length apoLp-III molecule offers the possibility of relating the structure of an apolipoprotein to its function. Because apoLp-III shares many physical-chemical properties with exchangeable apolipoproteins of humans and other vertebrates (1,(11)(12), it is likely that elucidation of the mechanism of interaction of apoLp-III with lipids would be useful to the understanding of the function of human apolipoproteins.Solving the structure of apolipoproteins in the lipid...

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Lipid-bound apoLp-III is less effective in binding to lipopolysaccharides and phosphatidylglycerol vesicles compared to the lipid-free protein

Wijeratne

Weers

2019

Mol Cell Biochem

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An N-Terminal Three-Helix Fragment of the Exchangeable Insect Apolipoprotein Apolipophorin III Conserves the Lipid Binding Properties of Wild-Type Protein

Cited by 19 publications

References 41 publications

Deletion of the N- or C-Terminal Helix of Apolipophorin III To Create a Four-Helix Bundle Protein

Deletion of the N- or C-Terminal Helix of Apolipophorin III To Create a Four-Helix Bundle Protein

Structure of Apolipophorin-III in Discoidal Lipoproteins

Lipid-bound apoLp-III is less effective in binding to lipopolysaccharides and phosphatidylglycerol vesicles compared to the lipid-free protein

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