Contributed by S. Walter Englander, December 19, 2016 (sent for review October 24, 2016; reviewed by Carl Frieden and Robert O. Ryan)
Apoliprotein E (apoE) serves as a cholesterol transport protein in both the peripheral circulation and the brain. In humans, the less common apoE4 isoform, which differs from the most abundant parent apoE3 by a single C112R substitution, is associated with increased incidence of cardiovascular disease and Alzheimer’s disease. To understand the structural basis for the altered functionality, we used hydrogen exchange mass spectrometry to compare the structure, stability, and molecular dynamics of these isoforms. The C112R substitution in apoE4 leads to unfolding of certain helical segments that reduces self-association and is expected to enhance the binding of apoE4 to triglyceride-rich lipoprotein particles in plasma and to amyloid-β deposits in the brain.
Apolipoprotein E (apoE) plays a critical role in cholesterol transport in both peripheral circulation and brain. Human apoE is a polymorphic 299-residue protein in which the less common E4 isoform differs from the major E3 isoform only by a C112R substitution. ApoE4 interacts with lipoprotein particles and with the amyloid-β peptide, and it is associated with increased incidence of cardiovascular and Alzheimer’s disease. To understand the structural basis for the differences between apoE3 and E4 functionality, we used hydrogen−deuterium exchange coupled with a fragment separation method and mass spectrometric analysis to compare their secondary structures at near amino acid resolution. We determined the positions, dynamics, and stabilities of the helical segments in these two proteins, in their normal tetrameric state and in mutation-induced monomeric mutants. Consistent with prior X-ray crystallography and NMR results, the N-terminal domain contains four α-helices, 20 to 30 amino acids long. The C-terminal domain is relatively unstructured in the monomeric state but forms an α-helix ∼70 residues long in the self-associated tetrameric state. Helix stabilities are relatively low, 4 kcal/mol to 5 kcal/mol, consistent with flexibility and facile reversible unfolding. Secondary structure in the tetrameric apoE3 and E4 isoforms is similar except that some helical segments in apoE4 spanning residues 12 to 20 and 204 to 210 are unfolded. These conformational differences result from the C112R substitution in the N-terminal helix bundle and likely relate to a reduced ability of apoE4 to form tetramers, thereby increasing the concentration of functional apoE4 monomers, which gives rise to its higher lipid binding compared with apoE3.
Author contributions: P.S.C., L.M., S.L.-K., S.W.E., and M.C.P. designed research; P.S.C. and S.L.-K. performed research; L.M., S.W.E., and M.C.P. analyzed data; and S.W.E. and M.C.P. wrote the paper.
Reviewers: C.F., Washington University School of Medicine; and R.O.R., Children's Hospital Oakland Research Institute.
The authors declare no conflict of interest.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1617523114/-/DCSupplemental.
