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Replacement of helix 1′ enhances the lipid binding activity of apoE3 N‐terminal domain
Author(s) -
Redmond Katherine A.,
Murphy Conrad,
Narayanaswami Vasanthy,
Kiss Robert S.,
Hauser Paul,
Guigard Emmanuel,
Kay Cyril M.,
Ryan Robert O.
Publication year - 2006
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2005.05089.x
Subject(s) - guanidine , helix (gastropod) , denaturation (fissile materials) , apolipoprotein b , chemistry , biochemistry , mutant , ldl receptor , lipoprotein , helix bundle , biophysics , conformational change , wild type , triple helix , protein structure , biology , stereochemistry , cholesterol , gene , ecology , nuclear chemistry , snail
The N‐terminal domain of human apolipoprotein E (apoE‐NT) harbors residues critical for interaction with members of the low‐density lipoprotein receptor (LDLR) family. Whereas lipid free apoE‐NT adopts a stable four‐helix bundle conformation, a lipid binding induced conformational adaptation is required for manifestation of LDLR binding ability. To investigate the structural basis for this conformational change, the short helix connecting helix 1 and 2 in the four‐helix bundle was replaced by the sequence NPNG, introducing a β‐turn. Recombinant helix‐to‐turn (HT) variant apoE3‐NT was produced in Escherichia coli , isolated and characterized. Stability studies revealed a denaturation transition midpoint of 1.9 m guanidine hydrochloride for HT apoE3‐NT vs. 2.5 m for wild‐type apoE3‐NT. Wild‐type and HT apoE3‐NT form dimers in solution via an intermolecular disulfide bond. Native PAGE showed that reconstituted high‐density lipoprotein prepared with HT apoE3‐NT have a diameter in the range of 9 nm and possess binding activity for the LDLR on cultured human skin fibroblasts. In phospholipid vesicle solubilization assays, HT apoE3‐NT was more effective than wild‐type apoE3‐NT at inducing a time dependent decrease in dimyristoylphosphatidylglycerol vesicle light scattering intensity. In lipoprotein binding assays, HT apoE3‐NT protected human low‐density lipoprotein from phospholipase C induced aggregation to a greater extent that wild‐type apoE3‐NT. The results indicate that a mutation at one end of the apoE3‐NT four‐helix bundle markedly enhances the lipid binding activity of this protein. In the context of lipoprotein associated full‐length apoE, increased lipid binding affinity of the N‐terminal domain may alter the balance between receptor‐active and ‐inactive conformational states.