Apolipoprotein E (apoE) is a major lipoprotein in the brain involved in Cholesterol transport and lipid metabolism.1, 2 There are three major isoforms of apoE, viz., apoE2 (Cys112, Cys158), apoE3 (Cys112, Arg158), and apoE4 (Arg112, Arg158)3 that differ by single amino acid substitutions. Since apoE4 was found to be associated with increased susceptibility to Alzheimer's disease (AD) and other neurodegenerative conditions there has been a surge of attention on the isoforms of apoE.4, 5, 6, 7, 8 While the molecular basis for the pathological role of apoE4 remains unclear, it is well-established that apoE4 is less effective in several key functions typically performed by apoE. For example, apoE2 and apoE3 have a higher binding preference for high-density lipoprotein (HDL) particles, whereas apoE4 exhibits a greater affinity for low-density lipoprotein (LDL) and very-low-density lipoprotein (VLDL) particles.9, 10, 11, 12, 13 Expression of apoE2 or apoE3 promotes protection and repair of the damaged neurons in response to stress or injury, but apoE4 is found to be less effective.2, 14 ApoE4 is also associated with poor outcomes in maintaining synaptic integrity and promoting Amyloid-β (Aβ) clearance in the brain.15, 16, 17, 18 ApoE4 is also more susceptible to intra-neuronal proteases, which generate neurotoxic fragments that escape the secretory pathways and cause mitochondrial dysfunction and alternations in the cytoskeleton.19 Despite the involvement of apoE4 in the pathology of multiple debilitating diseases the structural differences between the apoE isoforms still remains unknown.

The structure of full-length apoE (FL-apoE) consists of an N-terminal domain (NTD, residues: 1–167) and a C-terminal domain (CTD, residues: 191–299) connected by a hinge region.20 The structure of NTD, which harbors the residues 112 and 158, consists of a four-helix bundle that is found to be almost identical across the isoforms of apoE.21, 22 The CTD is believed to be partly helical and partly unstructured.23, 24, 25 While the structure of the full-length wild type (WT) apoE isoforms is not yet known, extensive research from Weisgraber and colleagues suggests that the apoE isoforms differ in terms of domain interactions.20, 26 It is hypothesized that apoE4 harbors a putative salt bridge between R61 and E255 connecting the NTD and the CTD.27, 28 This salt bridge and hence the domain interaction is proposed to be absent in apoE3 and apoE2. The inter-domain interaction in apoE4 is believed to markedly affect the physiological functions of apoE4. For example, apoE2 and apoE3 preferentially associate with the HDL particles in the periphery, but apoE4 is associated with LDL and VLDL.10, 12 However, direct evidence of the presence of the putative salt bridge in apoE4 is still lacking due to lack of a high resolution structure of this isoform. More importantly, biophysical characterization of the domain-domain interactions in apoE4 has not yet been done. Recently, Chen et al., published the solution NMR structure of the full-length but a monomeric mutant of apoE3, which exhibits multiple salt bridges and H-bonds between the resdiues in the NTD and CTD indicating presence of domin-domain interactions in apoE3.23 Since the structure of the monomeric mutant of apoE4 is still unknown, the isoform-specific differences in domain interaction remain unclear.

In this article, we examine the domain-domain interactions in apoE3 and apoE4 using Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS). Since the WT-apoE exhibits monomer-tetramer equilibrium, which complicates the interpretation of the HDX-MS protection factors, we have used the monomeric mutant of the full-lnegth (FL) apoE3 and apoE4.29, 30 To examine domain interactions we compare the kinetics of HDX of the FL-apoE proteins and the individual domain fragments, viz, the N-terminal fragment (NTF) and the C-terminal fragment (CTF). We observed that the HDX kinetics of the FL-apoE differ from that of the domain fragments, i.e., NTF-apoE + CTF-apoE, indicating interactions between the domains in the FL protein. Then, we identified the peptide fragments affected by the interdomain interactions in both the isoforms. Taken together, our results indicate that the presence of the CTD induces more extensive changes in the accessible conformational ensemble of apoE4 than apoE3.