Abstract 586: Ordered Opening of the 4-Helix Bundle in the LDL-Receptor Binding Domain of Human Apolipoprotein E3 as Revealed by Fluorescence Polarization Analysis

Abstract

Apolipoprotein E3 (apoE3) is an anti-atherogenic protein that helps maintain triglycerides and cholesterol levels in the plasma. It is responsible for binding and cellular uptake of plasma lipoproteins via the low-density lipoprotein receptor (LDLr) family of proteins. It is a highly alpha-helical protein that can exist in lipid-free and lipid-bound states, and undergoes a dramatic conformational re-organization when transitioning between the two states. The objective of this study is to understand the mechanism of chemical-induced unfolding of the 4-helix bundle N-terminal (NT) domain housing the LDLr binding sites. The rationale is that the ease of unfolding is a reflection of ease of helix bundle opening during lipid interaction. We tested the hypothesis that the 4-helix bundle undergoes a concerted opening to reveal the hydrophobic interior, analogous to opening an umbrella. Using X-ray and NMR structure guided rational approach, single cysteine-containing constructs were designed; a fluorescent probe was attached to the free -SH groups to serve as a spectroscopic reporter about the microenvironment and mobility of selected segments of the protein: the 4 main helices observed in the X-ray structure (H1 to H4) and the relatively flexible helixes flanking the helix bundle as observed in the NMR structure. We induced unfolding by chemical denaturation and monitored changes in protein secondary structure and tertiary conformation by circular dichroism and fluorescence spectroscopy, respectively. The overall global was relatively unaltered in the labeled variants; however, fluorescence emission and polarization studies revealed that during unfolding, the microenvironment of the probe on H3 and H4 is more polar and that these helices become more mobile at lower denaturant concentrations, compared to probes on H1 and H2. The data suggest that the 4-helix bundle undergoes a sequential unfolding pattern of the main helices in the following order: helix 4/helix 3, helix 2 and lastly helix 1. Our results offer insights into the mechanism of lipid and lipoprotein binding of apoE3 NT domain, with the segment towards the C-terminal end likely triggering lipid interaction.