Effects of lipid interaction on the lysine microenvironments in apolipoprotein E.
Lysines in apolipoprotein (apo) E are key factors in the binding of apoE to the low density lipoprotein receptor, and high affinity binding requires that apoE be associated with lipid. To gain insight into this effect, we examined the microenvironments of the eight lysines in the 22-kDa fragment of apoE3 (residues 1-191) in the lipid-free and lipid-associated states. As shown by (1)H,(13)C heteronuclear single quantum coherence nuclear magnetic resonance, lysine resonances in the lipid-free fragment were poorly resolved over a wide pH range, whereas in apoE3.dimyristoyl phosphatidylcholine (DMPC) discs, the lysine microenvironments and protein conformation were significantly altered. Sequence-specific assignments of the lysine resonances in the spectrum of the lipidated 22-kDa fragment were made. In the lipid-free protein, six lysines could be resolved, and all had pK(a) values above 10. In apoE3.DMPC complexes, however, all eight lysines were resolved, and the pK(a) values were 9.2-11.1. Lys-143 and Lys-146, both in the receptor binding region in helix 4, had unusually low pK(a) values of 9.5 and 9.2, respectively, likely as a result of local increases in positive electrostatic potential with lipid association. Shift reagent experiments with potassium ferricyanide showed that Lys-143 and Lys-146 were much more accessible to the ferricyanide anion in the apoE3.DMPC complex than in the lipid-free state. The angle of the nonpolar face of helix 4 is smaller than the angles of helices 1, 2, and 3, suggesting that helix 4 cannot penetrate as deeply into the DMPC acyl chains at the edge of the complex and that its polar face protrudes from the edge of the disc. This increased exposure and the greater positive electrostatic potential created by interaction with DMPC may explain why lipid association is required for high affinity binding of apoE to the low density lipoprotein receptor.
Lund-Katz
S
S
Joseph Stokes Jr. Research Institute, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104-4318, USA. katzs@email.chop.edu
Zaiou
M
M
Wehrli
S
S
Dhanasekaran
P
P
Baldwin
F
F
Weisgraber
K H
KH
Phillips
M C
MC
D001057:Apolipoproteins E / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D050356:Lipid Metabolism; D008239:Lysine / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D011487:Protein Conformation
D001057:Apolipoproteins E; D008239:Lysine