Human apolipoprotein (apo) E contains an amino-and a carboxyl-terminal domain, which are connected by a hinge region (approximately residues 165 to 215). The interaction of the two domains has been suggested to be responsible for the apoE4-binding preference for very low density lipoproteins (VLDL). In the absence of this interaction in apoE3, the preference is for high density lipoproteins (HDL). To exclude the possibility that the interaction of apoE with other apolipoproteins on the native

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... rticles may contribute to the isoform-specific preferences, VLDL-like emulsion particles were incubated with apoE, and the lipid-bound apoE was separated from free apoE on a Superose 6 column. The apoE4 bound more effectively to these particles than did apoE3, indicating that the apoE4 preference for VLDL is due not to interactions with other apolipoproteins but to an intrinsic property of apoE4, likely related to domain interaction. Previously, arginine 61 was shown to be critical for the isoform preferences, suggesting that it interacted with an acidic residue(s) in the carboxyl terminus. Substitution of arginine 61 with lysine did not alter the preference of apoE4 for VLDL, demonstrating that a positive charge rather than a specific requirement for arginine is critical for domain interaction. To identify the acidic residue(s) in the carboxyl terminus interacting with arginine 61, the six acidic residues (244, 245, 255, 266, 270, and 271) in a region known to be important for both lipoprotein association and isoformspecific preferences were substituted individually with alanine in apoE4. Only substitution of glutamic acid 255 altered the preference of apoE4 from VLDL to HDL, indicating that this was the sole residue in the carboxyl terminus that interacts with arginine 61. The participation of the hinge region in domain interaction was examined with internal deletion mutants. Deletion of the residues 186 -202 or 186 -223, representing major portions of the hinge region, had no effect on the apoE4 preference for VLDL. This suggests that the hinge region may act as a spacer that connects the two domains. Further deletion into the carboxyl-terminal domain (to residue 244) results in a loss of apoE4 VLDL binding. These studies establish that interaction of arginine 61 and glutamic acid 255 mediates apoE4 domain interaction. Apolipoprotein (apo) 1 E (299 amino acids) plays an important role in lipoprotein metabolism through its interaction with the low density lipoprotein (LDL) receptor (1, 2). The three common isoforms of apoE (apoE2, apoE3, and apoE4) are distinguished from each other by their cysteine/arginine content at two polymorphic sites. Apolipoprotein E3 contains cysteine and arginine at positions 112 and 158, respectively, whereas apoE2 contains cysteine and apoE4 contains arginine at both sites (3) . Apolipoprotein E4 is associated with higher plasma cholesterol and LDL concentrations than apoE3 and apoE2 and, as a result, is a risk factor for cardiovascular disease (4 -8). One of the distinct metabolic characteristics of apoE4 is its preferential association with very low density lipoproteins (VLDLs) (9 -11), which may contribute to elevated cholesterol and LDL levels (4, 11). In addition to its role in lipoprotein metabolism, apoE plays an important role in neurobiology (12, 13), the apoE4 allele being a major risk factor for Alzheimer's disease (14 -16). Apolipoprotein E contains two structural domains, which are connected by a hinge region (approximately residues 165 to 215) (17, 18). The amino-terminal domain (residues 1-191, 22-kDa fragment) contains the receptor-binding region (19 -21), and the carboxyl-terminal domain (residues 216 -299, 10-kDa fragment) contains the major lipid-binding determinants (11, (22) (23) (24) . Although the two domains are independently folded, each can influence the properties of the other (11, 25, 26), a concept referred to as domain interaction (2). For example, the substitution of cysteine 112 by arginine in the aminoterminal domain of apoE4 influences the lipid-binding property of the carboxyl-terminal domain and results in the preference of apoE4 for VLDL (11). In the absence of this interaction, as is the case with apoE3, the preference is for high density lipoproteins (HDL). The domain interaction in apoE4 requires the positive charge at position 112 (11). However, this charge does not interact directly with the carboxyl-terminal domain (22) ; rather, it changes the conformation of the arginine 61 side chain compared with that in apoE3 (22). Replacement of the arginine 61 with threonine shifts the lipoprotein preference of apoE4 from VLDL to HDL (22). This result suggests two possibilities: 1) the change in the conformation of the arginine 61 side chain results in an interaction of apoE4 with other apolipoproteins on VLDL particles, accounting for VLDL preference; or 2) arginine 61, as a key mediator of intramolecular domain interaction in apoE4, interacts with one or more acidic residues in the carboxyl terminus, probably by salt bridge formation. To distinguish between these possibilities, VLDL-like emulsion particles were used to examine the relative affinities of