Poster Presentation The 43rd Lorne Conference on Protein Structure and Function 2018

Oligomerisation and structural dynamics: New insights into native glycosylated apolipoprotein D (#150)

Claudia S. Kielkopf 1 2 , Jason K.K. Low 3 , Madhubrata Ghosh 4 , Y Mok 5 , Aaron Oakley 6 , Ganesh S Anand 4 , Brett Garner 1 2 , Simon H.J. Brown 1 2
  1. Illawarra Health and Medical Research Institute, Wollongong, NSW, Australia
  2. School of Biology, University of Wollongong, Wollongong, NSW, Australia
  3. School of Life and Environmental Sciences, University of Sydney, Sydney, NSW, Australia
  4. Department of Biological Science, National University of Singapore, Singapore
  5. Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Melbourne, Vic, Australia
  6. School of Chemistry, University of Wollongong, Wollongong, NSW, Australia

Human apolipoprotein (apoD) is a glycosylated 25 kDa lipocalin that folds into an eight-stranded beta-barrel with an adjacent alpha-helix (1). ApoD specifically binds small hydrophobic molecules such as progesterone and arachidonic acid (2). Furthermore, apoD reduces peroxidised lipids, which leads to dimerisation of apoD (3). Cerebral apoD is protective in Alzheimer’s disease (AD) through antioxidant function and attenuation of amyloid-beta pathology (4, 5). Oligomerisation is a common lipocalin feature and the apoD-homologue in insects, lazarillo, oligomerises (6, 7). Recombinant apoD from E.coli, however, is monomeric (8) and the oligomeric status of native apoD is unknown. Furthermore, it is unclear how apoD quaternary structure and dynamics change upon ligand binding.

Here, we investigated self-association of native, human apoD from plasma, cerebrospinal fluid (CSF) and breast cyst fluid (BCF) and glycosylated recombinant apoD. Native PAGE revealed apoD oligomers in BCF and CSF and in purified recombinant apoD. In contrast, apoD in plasma is associated high-density lipoprotein forming a high molecular weight complex (>900 kDa). BCF-purified apoD was characterised using size-exclusion chromatography (SEC), lysine-crosslinking, analytical ultracentrifugation (AUC), multi-angle laser-light scattering (MALLS) and SEC-small-angle X-ray scattering (SEC-SAXS). These techniques consistently showed that the predominant apoD form in BCF is an ~100 kDa tetramer which dissociates upon dilution. SAXS provided a structural model of a glycosylated apoD tetramer.

Hydrogen-deuterium exchange MS (HDX-MS) was utilised to probe apoD dynamics in the apo- and progesterone-bound state by quantification of deuteron exchange in backbone amides. Achieving ~85% coverage, we show that upon progesterone-binding, the overall structure of apoD becomes more rigid, potentially mediated by progesterone-binding. We observe orthosteric changes in the binding pocket and allosteric changes in distal apoD regions, suggesting communication of progesterone-binding across the structure. Our data reveal new insights into apoD structure and oligomerisation that may eventually help to explain its protective role in AD.

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