Lipid association-induced N- and C-terminal domain reorganization in humanapolipoprotein E3

Apolipoprotein E (apoE) is a 299 amino acid, antiatherogenic protein that p lays a key role in regulating plasma lipoprotein metabolism. It is composed of an N-terminal (NT) domain (residues 1-191) that is responsible for bind ing to members of the low density lipoprotein receptor family and a C-termi nal (CT) domain (residues 216-299) that anchors the protein to lipoprotein particles by virtue of its high-affinity lipid binding characteristics. Iso form-specific differences in the NT domain that modulate the lipoprotein bi nding preference elicited by the CT domain suggest the existence and import ance of domain interactions in this protein. Employing steady state fluores cence quenching and resonance energy transfer techniques, spatial proximity relationships between the N- and C-terminal domains were investigated in r ecombinant human apoE3. ApoE3 containing a single Trp at position 264 and a n N-iodoacetyl-N'-(5-sulfo-1-napthyl) ethylenediamine (AEDANS) moiety coval ently attached to the lone Cys residue at position 112 was used (AEDANS-apo E3/W@264). Fluorescence quenching studies revealed a solvent-exposed locati on for Trp-264. In the lipid-free state, fluorescence resonance energy tran sfer (FRET) was noted between Trp-264 and AEDANS, with a calculated distanc e of 27 Angstrom between the two fluorophores. Control experiments establis hed that FRET observed in this system is intramolecular. FRET was abolished upon proteolysis in the linker region connecting the NT and CT domains. Lo wering the solution pH to 4 induced an increase in the efficiency of intram olecular energy transfer, with the two domains reorienting about 5 Angstrom closer to one another. Interdomain FRET was retained in the presence of 0. 6-1.0 M guanidine hydrochloride but was lost at higher concentrations, a ma nifestation of unfolding of the domains and increased distance between the donor-acceptor pair. Interaction of AEDANS-apoE3/W@264 with lipid induced a loss of FRET, attributed to spatial repositioning of the domains by > 80 A ngstrom. The data provide biophysical evidence that, in addition to reporte d conformational changes in the four-helix bundle configuration induced by lipid association, lipid binding of apoE is accompanied by reorientation of the tertiary disposition of the NT and CT domains.