Identification of a novel tetrameric structure for human apolipoprotein-D

Kielkopf, Claudia S.; Low, Jason K. K.; Mok, Yee-Foong; Bhatia, Surabhi; Palasovski, Tony; Oakley, Aaron J.; Whitten, Andrew E.; Garner, Brett; Brown, Simon H. J.

doi:10.1101/335232

Cited by 3 publications

(11 citation statements)

References 57 publications

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“…Western-Blot analysis using anti-GFP antibodies revealed that an approximately 50 kDa PfLCN-GFP (calculated MW 50.8 kDa) was not only found in the soluble fraction but also in the sodium carbonate and Triton fraction, indicating that it is a soluble protein but that it can also associate with and integrate into membranes ( Figure 3D). Interestingly, we also detected a higher molecular mass GFP-positive band, which likely represents PfLCN in its dimeric state as previously described for other lipocalins (Bhatia et al, 2012;Van Veen et al, 2006;Kielkopf et al, 2018) and as also supported by our SAXS analysis. In addition to these bands, a third band at around 25 kD was visible that likely represents cleaved GFP.…”

Section: Pflcn Localizes To the Pv And Fv And Can Integrate Into Membsupporting

confidence: 90%

“…In addition, it may indicate that PfLCN can also dimerize in vivo. In this respect it is interesting to note that reducing SDS-PAGE resistant dimers have already been observed for other lipocalins (Bhatia et al, 2012;Van Veen et al, 2006;Kielkopf et al, 2018).…”

Section: Discussionmentioning

confidence: 69%

“…This observation excludes that tetramerization is an artefact of the crystallization process. Oligomerization is a common feature of lipocalins and often occurs upon ligand binding (Kielkopf et al, 2018). The fact that PfLCN was in an oligomeric state in the crystal structure and in solution without a ligand bound suggests that the multimerization of PfLCN occurs independently of its ligand.…”

Section: Discussionmentioning

confidence: 99%

“…When we compared the structure of PfLCN to human members of the lipocalin family, it became evident that PfLCN shows high structural similarity to human ApoD. Interestingly, the tetramerization of ApoD under physiological condition has recently been described (Kielkopf et al, 2018). Human ApoD is capable of binding to a range of small hydrophobic ligands such as progesterone and arachidonic acid and exerts an antioxidant function, which is partially due to the reduction of peroxidized lipids (Bhatia et al, 2012).…”

Section: Discussionmentioning

confidence: 99%

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Structure-based identification and functional characterization of an essential lipocalin in the malaria parasitePlasmodium falciparum

Burda

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Lauk

et al. 2020

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Proteins of the lipocalin family are known to bind small hydrophobic ligands and are involved in various physiological processes ranging from lipid transport to oxidative stress responses. The genome of the malaria parasite Plasmodium falciparum contains a single protein PF3D7_0925900 with a lipocalin signature. Using crystallography and small-angle X-ray scattering, we show that the protein has a tetrameric structure of typical lipocalin monomers, hence we name it P. falciparum lipocalin (PfLCN), the first lipocalin structurally and functionally characterized in a single-celled eukaryote. We show that PfLCN is expressed in the intraerythrocytic stages of the parasite and localizes to the parasitophorous and food vacuoles. Conditional knockdown of PfLCN impairs parasite development, which can be rescued by treatment with the radical scavenger Trolox or by temporal inhibition of hemoglobin digestion. This suggests a key function of PfLCN in counteracting oxidative stress induced cell damage during multiplication of parasites within red blood cells.Keywords: blood stage / food vacuole / lipocalin / malaria / oxidative stress compound 2. Images and time-lapse movies were acquired on microscopes of the CSSB imaging facility.

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Section: Pflcn Localizes To the Pv And Fv And Can Integrate Into Membsupporting

confidence: 90%

Section: Discussionmentioning

confidence: 69%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Structure-based identification and functional characterization of an essential lipocalin in the malaria parasitePlasmodium falciparum

Burda

Crosskey

Lauk

et al. 2020

Preprint

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“…ApoD has an antioxidant function and plays a protective role in Alzheimer's disease [6,7]. 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].…”

Section: Introductionmentioning

confidence: 99%

Small angle X-ray scattering analysis of ligand-bound forms of tetrameric apolipoprotein-D

et al. 2021

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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.

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HDX‐MS reveals orthosteric and allosteric changes in apolipoprotein‐D structural dynamics upon binding of progesterone

et al. 2018

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Apolipoprotein-D is a glycosylated tetrameric lipocalin that binds and transports small hydrophobic molecules such as progesterone and arachidonic acid. Like other lipocalins, apolipoprotein-D adopts an eight-stranded β-barrel fold stabilized by two intramolecular disulphide bonds, with an adjacent α-helix. Crystallography studies of recombinant apolipoprotein-D demonstrated no major conformational changes upon progesterone binding. Amide hydrogen-deuterium exchange mass spectrometry (HDX-MS) reports structural changes of proteins in solution by monitoring exchange of amide hydrogens in the protein backbone with deuterium. HDX-MS detects changes in conformation and structural dynamics in response to protein function such as ligand binding that may go undetected in X-ray crystallography, making HDX-MS an invaluable orthogonal technique. Here, we report an HDX-MS protocol for apolipoprotein-D that solved challenges of high protein rigidity and low pepsin cleavage using rigorous quenching conditions and longer deuteration times, yielding 85% sequence coverage and 50% deuterium exchange. The relative fractional deuterium exchange of ligand-free apolipoprotein-D revealed apolipoprotein-D to be a highly structured protein. Progesterone binding was detected by significant reduction in deuterium exchange in eight peptides. Stabilization of apolipoprotein-D dynamics can be interpreted as a combined orthosteric effect in the ligand binding pocket and allosteric effect at the N-terminus and C-terminus. Together, our experiments provide insight into apolipoprotein-D structural dynamics and map the effects of progesterone binding that are relayed to distal parts of the protein. The observed stabilization of apolipoprotein-D dynamics upon progesterone binding demonstrates a common behaviour in the lipocalin family and may have implications for interactions of apolipoprotein-D with receptors or lipoprotein particles. Statement: We reveal for the first time how apolipoprotein-D, which is protective in Alzheimer's disease, becomes more ordered when bound to a molecule of steroid hormone. These results significantly extend the understanding of apolipoprotein-D structure from X-ray crystallography studies by

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Identification of a novel tetrameric structure for human apolipoprotein-D

Cited by 3 publications

References 57 publications

Structure-based identification and functional characterization of an essential lipocalin in the malaria parasitePlasmodium falciparum

Structure-based identification and functional characterization of an essential lipocalin in the malaria parasitePlasmodium falciparum

Small angle X-ray scattering analysis of ligand-bound forms of tetrameric apolipoprotein-D

HDX‐MS reveals orthosteric and allosteric changes in apolipoprotein‐D structural dynamics upon binding of progesterone

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