Mechanism of lipid binding of human apolipoprotein E3 by hydrogen/deuterium exchange/mass spectrometry and fluorescence polarization

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 proteinprotein interaction in this segment. functional assays showed that locking apoe(201-299) in an inter-molecular disulfidebonded 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. conclusion: 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. © 2016 bentham science publishers.