A Monomeric, Biologically Active, Full-Length Human Apolipoprotein E

Abstract

Apolipoprotein E (apoE) is an exchangeable apolipoprotein that plays an important role in lipid/lipoprotein metabolism and cardiovascular diseases. Recent evidence indicates that apoE is also critical in several other important biological processes, including Alzheimer's disease, cognitive function, immunoregulation, cell signaling, and infectious diseases. Although the X-ray crystal structure of the apoE N-terminal domain was solved in 1991, the structural study of full-length apoE is hindered by apoE's oligomerization property. Using protein-engineering techniques, we generated a monomeric, biologically active, full-length apoE. Cross-linking experiments indicate that this mutant is nearly 95-100% monomeric even at 20 mg/mL. CD spectroscopy and guanidine hydrochloride denaturation demonstrate that the structure and stability of the monomeric mutant are identical to wild-type apoE. Monomeric and wild-type apoE display similar lipid-binding activities in dimyristoylphosphatidylcholine clearance assays and formation of reconstituted high-density lipoproteins. Furthermore, the monomeric and wild-type apoE proteins display an identical LDL receptor binding activity. Availability of this monomeric, biologically active, full-length apoE allows us to collect high quality NMR data for structural determination. Our initial NMR data of lipid-free apoE demonstrates that the N-terminal domain in the full-length apoE adopts a nearly identical structure as the isolated N-terminal domain, whereas the C-terminal domain appears to become more structured than the isolated C-terminal domain fragment, suggesting a weak domain-domain interaction. This interaction is confirmed by NMR examination of a segmental labeled apoE, in which the N-terminal domain is deuterated and the C-terminal domain is double-labeled. NMR titration experiments further suggest that the hinge region (residues 192-215) that connects apoE's N- and C-terminal domains may play an important role in mediating this domain-domain interaction.

Helical structure, stability, and dynamics in human apolipoprotein E3 and E4 by hydrogen exchange and mass spectrometry

“…Cleavage with thrombin leaves the target apoE with two extra amino acids, glycine and serine (designated residues -2, -1), at the N terminus that do not significantly alter the properties of the protein. The QuikChange site-directed mutagenesis kit (Stratagene) was used to introduce the mutations F257A/W264R/V269A/L279Q/V287E into the C-terminal domain and generate monomeric variants of apoE3 and apoE4 (14). The apoE preparations were at least 95% pure as assessed by SDS/PAGE.…”

“…A highresolution structure of the wild-type (WT) apoE molecule is unavailable because both apoE3 and E4 self-associate through their C-terminal domains to form a tetramer, which has inhibited study by X-ray crystallography and NMR. It has been possible to obtain the structures of the N-and C-terminal domains using a monomeric variant with five mutations (F257A/W264R/V269A/L279Q/V287E) in the C-terminal domain that interfere with tetramer formation while maintaining similar lipid-binding activity (14,15). NMR study of the monomeric variant of human apoE3 (16) and crystallography of the isolated N-terminal domain (1,17) show that residues 23-167 form an N-terminal antiparallel four-helix bundle.…”

“…There also may be an intramolecular contribution to be considered. Thus, in the monomeric state, the N-and C-terminal domains interact (14), and it is possible that modified intramolecular domain−domain interaction (and length of hinge region) in apoE4 compared with apoE3 ( Fig. 6) also contributes to the alteration in structure of the tetramer.…”

Structural differences between apolipoprotein E3 and E4 as measured by 19F NMR

“…Since the wild-type proteins are oligomers of the 34 kDa monomer, the monomeric forms of apoE were prepared following the procedure of Zhang et al 10 who showed that by making four to five mutations in the C-terminal region a monomeric form of apoE3 could be obtained. Figure 3 shows the NMR spectra of both mutated apoE3 and mutated apoE4.…”

“…7 Recently two mutants involving four to five mutations in the C-terminal domain of apoE3 have been described which result in a protein that remains monomeric even at 25 mg/mL. 10 Using this mutation, Zhang et al have reported the complete NMR backbone assignment of monomeric apoE3. 11 Provided that these mutants are structurally similar to wild-type, they would be useful systems to examine the properties of the monomeric forms of these proteins.…”