title: Mechanism of Lipid Binding of Human Apolipoprotein E3 by Hydrogen/Deuterium Exchange/Mass Spectrometry and Fluorescence Polarization.
abstract: BACKGROUND
  Human apolipoprotein E3 (apoE3) is an exchangeable apolipoprotein that plays a critical role in maintaining plasma cholesterol/triglyceride homeostasis. The C-terminal (CT) domain of apoE3 (residues 201-299) is composed of amphipathic α-helices C1: W210-S223, C2: V236-E266, and C3: D271-W276, which play a dominant role in mediating high-affinity lipid binding.
  
  
  OBJECTIVE
  The objective is to understand the accessibility of the CT domain at the sub-domain level and the mechanistic details regarding lipid-binding interaction.
  
  
  METHODS
  Hydrogen-deuterium exchange coupled to mass spectrometry (HDX/MS) of recombinant wild type (WT) apoE(201-299), chemical-induced unfolding monitored as changes in fluorescence polarization (FP) of labeled apoE(201-299) bearing a probe at specified sites, and lipid binding studies were carried out.
  
  
  RESULTS
  HDX/MS revealed that residues towards the C-terminal end of the domain display significantly lower %D uptake compared to those towards the center, suggesting extensive protein-protein interaction in this segment. Functional assays showed that locking apoE(201-299) in an inter-molecular disulfide-bonded state at position 209, 223, 255, or 277 significantly decreases its ability to interact with lipids, especially when tethered towards the ends; this could be restored by reduction. Unfolding studies indicate that the C-terminal end offers less resistance to unfolding compared to the central portion of the domain.
  
  
  CONCLUSIONS
  Taken together, our data suggest that two dimers of CT domain are juxtaposed around helix C3 leading to apoE3 tetramerization, and that dissociation to monomeric units is a required step in lipid binding, with helix C3 likely seeking stability via lipid interaction prior to helices C1 or C2.
authors.forename: Charina S
authors.initials: CS
authors.lastname: Fabilane
authors.forename: Patricia N
authors.initials: PN
authors.lastname: Nguyen
authors.forename: Roy V
authors.initials: RV
authors.lastname: Hernandez
authors.forename: Sasidhar
authors.initials: S
authors.lastname: Nirudodhi
authors.forename: Mai
authors.initials: M
authors.lastname: Duong
authors.forename: Claudia S
authors.initials: CS
authors.lastname: Maier
authors.affiliation: Department of Chemistry & Biochemistry, 1250 Bellflower Blvd, California State University Long Beach, Long Beach, CA 90840, USA. vas.narayanaswami@csulb.edu.
authors.forename: Vasanthy
authors.initials: V
authors.lastname: Narayanaswami
mesh_terms: D053318:Apolipoprotein E3 / Q000737:chemistry* / Q000378:metabolism*; D001057:Apolipoproteins E / Q000737:chemistry* / Q000378:metabolism*; D041961:Deuterium Exchange Measurement; D005454:Fluorescence Polarization; D006801:Humans; D050356:Lipid Metabolism; D013058:Mass Spectrometry; D008958:Models, Molecular; D011485:Protein Binding; D055503:Protein Multimerization; D058767:Protein Unfolding; D013050:Spectrometry, Fluorescence
chemical_list: C000612345:ApoE protein, human; D053318:Apolipoprotein E3; D001057:Apolipoproteins E