Hydrogen/deuterium exchange and electron-transfer dissociation mass spectrometry determine the interface and dynamics of apolipoprotein E oligomerization

Abstract

Apolipoprotein E, a 34 kDa protein, plays a key role in triglyceride and cholesterol metabolism. Of the three common isoforms (ApoE2, -3, and -4), only ApoE4 is a risk factor for Alzheimer's disease. All three isoforms of wild-type ApoE self-associate to form oligomers, a process that may have functional consequences. Although the C-terminal domain, residues 216-299, of ApoE is believed to mediate self-association, the specific residues involved in this process are not known. Here we report the use of hydrogen/deuterium exchange (H/DX) coupled with enzymatic digestion to identify those regions in the sequence of full-length apoE involved in oligomerization. For this determination, we compared the results of H/DX of the wild-type proteins and those of monomeric forms obtained by modifying four residues in the C-terminal domain. The three wild-type and mutant isoforms show similar structures based on their similar H/DX kinetics and extents of exchange. Regions of the C-terminus (residues 230-270) of the ApoE isoforms show significant differences of deuterium uptake between oligomeric and monomeric forms, confirming that oligomerization occurs at these regions. To achieve single amino acid resolution, we examined the extents of H/DX by using electron transfer dissociation (ETD) fragmentation of peptides representing selected regions of both the monomeric and the oligomeric forms of ApoE4. From these experiments, we could identify the specific residues involved in ApoE oligomerization. In addition, our results verify that ApoE4 is composed of a compact structure at its N-terminal domain. Regions of C-terminal domain, however, appear to lack defined structure.

Figure 1

Peptide-level H/DX kinetics of ApoE4. A comparison between wild type (blue) and monomeric mutant (pink) shows significant differences in H/DX only for peptides 230–243 and 262–270 and small difference for peptide 271–279.

Figure 2

Comparison of H/DX kinetics of peptide 230–243 and 262–270 among all three isoforms (ApoE2, ApoE3, and ApoE4). An example of the mass spectra of peptide 262–270 of ApoE4 as a function of H/DX time is shown on the right (the red, dotted line is a reference to guide the eye).

Figure 3

A diagram of the H/DX-ETD procedure. A peptide of interest produced from on-line pepsin digestion is subjected to ETD fragmentation. Peptide coverage (~89%) of ApoE4 by applying this strategy is shown in right (four substitution sites to achieve the MM are marked in red).

Figure 4

ETD product-ion spectra of peptide 262–270 of ApoE4 after H/DX. The centroid of the distribution of c ions from monomeric mutant shows there are significant differences in the extent of H/DX between WT and MM for this region.

Figure 5

The extent of deuterium uptake of various amino-acid residues in the C-terminal domain of ApoE4 (Residue 220–299). Several residues of wild type (blue) have lower level (<0.3D) of deuterium uptake compared to the monomeric mutant (pink). Note: The gaps correspond to missing information.

Figure 6

The extent of deuterium uptake of each residue of ApoE4 monomeric mutant after correcting for back exchange (pink). Most of the residues in the C-terminus have high extent of deuterium uptake, indicating a flexible structure in this region. The extent of deuterium uptake in N-terminal region are consistent with the known secondary structure (top). (flexible loop, black line; flexible helix, gray box; rigid helix, black box). Note: The gaps correspond to missing information.

Figure 7

Structure of ApoE4 on which residue-level H/DX of ApoE4 monomeric mutant is mapped. Left: X-ray crystal structure of N-terminal region of ApoE4 (PDB: 1GS9). Right: Proposed secondary structure of C-terminal region of ApoE (5). Possible helices are shown in dotted boxes. Different colors represent different levels of deuterium uptake for each residue (lower left).