Abstract
Human apolipoprotein-D (apoD) is a glycosylated lipocalin that plays a protective role in Alzheimer’s disease due to its antioxidant function. Native apoD from human body fluids forms oligomers, predominantly a stable tetramer. As a lipocalin, apoD binds and transports small hydrophobic molecules such as progesterone, palmitic acid and sphingomyelin. Oligomerisation is a common trait in the lipocalin family and is affected by ligand binding in other lipocalins. The crystal structure of monomeric apoD shows no major changes upon progesterone binding. Here, we used small-angle X-ray scattering (SAXS) to investigate the influence of ligand binding and oxidation on apoD oligomerisation and conformation. As a solution-based technique, SAXS is well suited to detect changes in oligomeric state and conformation in response to ligand binding. Our results show no change in oligomeric state of apoD and no major conformational changes or subunit rearrangements in response to binding of ligands or protein oxidation. This highlights the highly stable structure of the native apoD tetramer under various physiologically relevant experimental conditions.
Highlights
Apolipoprotein-D is a ∼25 kDa glycoprotein belonging to the protein family of lipocalins [1,2], a family which is characterised by high structural homology and the ability to bind and transport small hydrophobic ligands [3]
Using small angle X-ray scattering (SAXS) and other techniques, we have previously shown that the apo form of native human apoD isolated from breast cyst fluid (BCF) forms a tetrameric oligomer that is stable upon dilution [8]
To evaluate if ligand binding or oxidation influences apoD oligomeric state or causes structural rearrangement of apoD subunits, apoD was incubated with ligands or oxidised using H2O2, and subjected to size exclusion chromatography (SEC)-SAXS analysis
Summary
Apolipoprotein-D (apoD) is a ∼25 kDa glycoprotein belonging to the protein family of lipocalins [1,2], a family which is characterised by high structural homology and the ability to bind and transport small hydrophobic ligands [3]. ApoD adopts a typical lipocalin fold of a β-barrel ligand pocket and an adjacent α-helix (Figure 1, PDB ID: 2HZQ). The crystal structure of apoD shows that upon progesterone binding, three side chains change conformation (Figure 1) [1]. Oligomerisation is common in the lipocalin family and has been shown in other lipocalins to be influenced by ligand binding, salt concentration and pH [3,9,10,11]. Ligand binding affects oligomerisation and vice versa in lipocalins [11,12]. Ligand binding of β-lactoglobulin leads to dimer dissociation [11], and ligand binding of crustacyanin, a pigmentation protein, is critically dependent on dimer formation [12]
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