Poster Presentation The 44th Lorne Conference on Protein Structure and Function 2019

When ligand meets protein: Structural characterisation of apolipoprotein-D during lipid interaction (#208)

Claudia S Kielkopf 1 2 , Jason KK Low 3 , Madhubrata Ghosh 4 , Y Mok 5 , Aaron Oakley 1 2 , Ganesh S Anand 6 , Brett Garner 1 2 , Simon HJ Brown 1 2
  1. Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong, NSW, Australia
  2. School of Chemistry and Molecular Bioscience, University of Wollongong, Wollongong, NSW, Australia
  3. School of Life and Environmental Sciences, University of Sydney, Sydney, NSW, Australia
  4. Institute of Bioengineering and Nanotechnology, 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. Department of Biological Science, National University of Singapore, Singapore

Proteins are structurally dynamic and their flexibility allows execution of protein functions, including interactions with other molecules. In fact, structural changes upon ligand-binding provide vital information about protein function. However, structural changes in response to ligand-binding can be challenging to detect with X-ray crystallography as crystallisation conditions may mask or suppress effects of ligand-binding, including oligomeric effects.1 We use a suite of structural and biochemical methods to reveal ligand-induced changes in structure and dynamics of native glycosylated apolipoprotein-D.

Apolipoprotein-D belongs to the lipocalin family and specifically binds and transports lipids such as progesterone and arachidonic acid in a hydrophobic binding pocket.2 Apolipoprotein-D can reduce peroxidised lipids3 and cerebral apolipoprotein-D is protective in Alzheimer’s disease through its antioxidant function and attenuation of amyloid-β pathology.4,5

The crystal structure of non-glycosylated, monomeric apolipoprotein-D revealed no major conformational changes upon progesterone-binding.6 Using chromatographic, scattering and ultracentrifugation techniques, we recently showed that native apolipoprotein-D purified from human breast cyst fluid forms a novel stable tetrameric complex.7 We use hydrogen-deuterium exchange mass spectrometry to probe native apolipoprotein-D dynamics in the apo and progesterone-bound state by quantification of deuterium exchange in backbone amides. Ligand-free apolipoprotein-D showed low deuterium exchange indicating a well-ordered protein structure with low dynamics. Progesterone-binding was detected through reduced orthosteric apolipoprotein-D dynamics in the binding pocket. We also observed allosteric changes in apolipoprotein-D dynamics at the N- and C-termini suggesting communication of progesterone-binding across the structure. Stabilisation of apolipoprotein-D dynamics upon progesterone-binding demonstrates a common behaviour in lipocalins and may have implications for interactions of apolipoprotein-D with receptors or lipoprotein particles. Our results significantly extend our knowledge about native apolipoprotein-D structure and protein-ligand interaction beyond the apolipoprotein-D crystal structure. We now use cryo-electron microscopy single-particle analysis aiming to provide further insights into the quaternary structure of apolipoprotein-D and potential additional changes upon ligand-binding.

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  7. Kielkopf CS, Low JKK, Mok YF, Bhatia S, Palasovski T, Oakley AJ, Whitten AE, Garner B, Brown SHJ. Identification of a novel tetrameric structure for human apolipoprotein-D. J Struct Biol 2018.