Lipid-induced extension of apolipoprotein E helix 4 correlates with low density lipoprotein receptor binding ability.
Apolipoprotein E (apoE) serves as a ligand for the low density lipoprotein receptor (LDLR) only when bound to lipid. The N-terminal domain of lipid-free apoE exists as globular 4-helix bundle that is conferred with LDLR recognition ability after undergoing a lipid binding-induced conformational change. To investigate the structural basis for this phenomenon, site-directed spin label electron paramagnetic resonance spectroscopy experiments were conducted, focusing on the region near the C-terminal end of helix 4 (Ala-164). Using C112S apoE-N-terminal as template, a series of single cysteine substitution variants (at sequence positions 161, 165, 169, 173, 176, and 181) were produced, isolated, and labeled with the nitroxide probe, methane thiosulfonate. Electron paramagnetic resonance analysis revealed that lipid association induced fixed secondary structure in a region of the molecule known to exist as random coil in the lipid-free state. In a complementary approach, site-directed fluorescence analysis using an environmentally sensitive probe indicated that the lipid-induced transition of this region of the protein to alpha helix was accompanied by relocation to a more hydrophobic environment. In studies with full-length apoE single Cys variants, a similar random coil to stable backbone transition was observed, consistent with the concept that lipid interaction induced an extension of helix 4 beyond the boundary defining its lipid-free conformation. This structural transition likely represents a key conformational change necessary for manifestation of the LDLR recognition properties of apoE.
Gupta
Vinita
V
Center for the Prevention of Obesity, Cardiovascular Disease and Diabetes, Children's Hospital Oakland Research Institute, Oakland, California 94609, USA.
Narayanaswami
Vasanthy
V
Budamagunta
Madhu S
MS
Yamamato
Taichi
T
Voss
John C
JC
Ryan
Robert O
RO
D020816:Amino Acid Motifs; D001057:Apolipoproteins E / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D008055:Lipids / Q000737:chemistry*; D009569:Nitric Oxide / Q000737:chemistry; D010743:Phospholipids / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011973:Receptors, LDL / Q000737:chemistry*; D013050:Spectrometry, Fluorescence
D001057:Apolipoproteins E; D008055:Lipids; D010743:Phospholipids; D011973:Receptors, LDL; D009569:Nitric Oxide; D003545:Cysteine