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

Abstract

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.apolipoprotein E | hydrogen exchange mass spectrometry | cholesterol | protein secondary structure | amphipathic helix

A mechanism for lipid binding to apoE and the role of intrinsically disordered regions coupled to domain–domain interactions

“…Both our HDX study and those of Chetty et al (22) support the validity of the NMR structure of the apoE3 monomeric mutant as determined by Chen et al (10). Any differences in comparing wild-type apoE3 and apoE4 between our data and those of Chetty et al (22) are likely due to different sample handling and data analysis methods.…”

Apolipoprotein E particle size is increased in Alzheimer's disease

“…Nuclear magnetic resonance [7] and hydrogen‐deuterium exchange mass spectrometry studies [10–12] also showed that Arg112 interacts with Lys95, leading to a marked change in protein folding. On the basis of these and other structural studies, phthalazinone derivatives known as “correctors” were designed in an attempt to inhibit this interaction and convert apoE4 to an E3‐like configuration [13].…”

Ordered opening of LDL receptor binding domain of human apolipoprotein E3 revealed by hydrogen/deuterium exchange mass spectrometry and fluorescence spectroscopy

“…A recent study of intact tetrameric apoE3 by HDX-MS [43] found the helix bundle organization to be broadly similar to that of the monomeric apoE3 studied by NMR. These authors noted a stable NT domain helix bundle and a long CT helix with markedly reduced stability for apoE3.…”

“…Interestingly, other researchers also reported that 27 out 43 peptides generated from the N-terminal segment (residues 1-191) of full length apoE3 elicited EX1/EX2 kinetics [43], indicating high occurrence of mixed HDX kinetics. Due to the challenges fitting mixed EX1/EX2 HDX kinetic mechanism, we classified (Figure S2) 15 peptides into two groups based on their experimentally observed HDX pattern: those that exhibit EX1/EX2 mixed HDX profile and those that exhibit binomial HDX profile (Table 1).…”