Oral Presentation The 45th Lorne Conference on Protein Structure and Function 2020

For when it’s complicated: How an integrative structural approach sheds light on native apolipoprotein-D structure (#30)

Claudia S Kielkopf 1 2 3 , Lance Wilson 4 , James C Bouwer 1 3 , Brett Garner 1 2 3 , Simon HJ Brown 1 3
  1. School of Chemistry and Molecular Bioscience, University of Wollongong, Wollongong, NSW, Australia
  2. Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong, NSW, Australia
  3. Molecular Horizons, University of Wollongong, Wollongong, NSW, Australia
  4. MASSIVE, Monash University, Melbourne, VIC, Australia

Integrative structural approaches have significantly advanced our understanding of molecular machines and large biological assemblies.1 The combined power of integrated techniques is also beneficial for challenging single proteins. We showcase the application of integrated structural approaches to elucidate the native oligomeric state and ligand-binding effects of the glycosylated lipocalin apolipoprotein-D. Apolipoprotein-D plays a protective role in Alzheimer’s disease through its role as a lipid anti-oxidant and lipid transporter. 2-4

Utilising non-recombinantly purified apolipoprotein-D, we showed with a suite of complementary techniques including MALLS that native apolipoprotein-D forms oligomers, predominantly a 100 kDa species consistent with a tetramer.5 This novel species is stable upon dilution and ligand-binding. Small-angle X-ray scattering (SAXS) revealed that ligand-binding does not cause large conformational changes. SAXS also provided a structural model of oligomeric apoD. Modelling was guided by molecular dynamics simulations of glycosylated monomeric apolipoprotein and restraints derived from crosslinking mass spectrometry.

To further probe ligand-binding effects, we measured apolipoprotein-D dynamics in the apo and progesterone-bound state using hydrogen-deuterium exchange mass spectrometry.6 Progesterone binding was detected through reduced 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 via hydrogen bond networks.

We currently employ single-particle cryo-electron microscopy to obtain a three-dimensional volume that allows fitting of the monomeric model. We face challenges in sample preparation and data analysis associated with a small protein of ~100 kDa including flexible glycosylations. These challenges are addressed by trialling grid types and data analysis strategies using multiple software packages. Best two-dimensional classes show secondary structures and potential side views resulting in preliminary three-dimensional volumes.

Our integrative approach overcomes ambiguity associated with a natively glycosylated protein and small protein size in cryo-EM and significantly advanced our understanding of native apolipoprotein-D structure and ligand-binding.

  1. 1. Ward AB, Sali A, Wilson IA. Integrative structural biology. Science 2013;339(6122):913-5.
  2. 2. Li H, Ruberu K, Muñoz SS, Jenner AM, Spiro A, Zhao H, Rassart E, Sanchez D, Ganfornina MD, Karl T and others. Apolipoprotein D modulates amyloid pathology in APP/PS1 Alzheimer's disease mice. Neurobiol Aging 2015;36(5):1820-33.
  3. 3. Breustedt DA, Schonfeld DL, Skerra A. Comparative ligand-binding analysis of ten human lipocalins. Biochim Biophys Acta 2006;1764(2):161-73.
  4. 4. Bhatia S, Knoch B, Wong J, Kim WS, Else PL, Oakley AJ, Garner B. Selective reduction of hydroperoxyeicosatetraenoic acids to their hydroxy derivatives by apolipoprotein D: implications for lipid antioxidant activity and Alzheimer's disease. The Biochemical journal 2012;442(3):713-21.
  5. 5. 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;203(3):205-218.
  6. 6. Kielkopf CS, Ghosh M, Anand GS, Brown SHJ. HDX-MS reveals orthosteric and allosteric changes in apolipoprotein-D structural dynamics upon binding of progesterone. Protein Sci 2019;28(2):365-374.