title: Replacement of helix 1' enhances the lipid binding activity of apoE3 N-terminal domain.
abstract: 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 beta-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.
authors.affiliation: Lipid Biology in Health and Disease Research Group, Children's Hospital Oakland Research Institute, CA 94609, USA.
authors.forename: Katherine A
authors.initials: KA
authors.lastname: Redmond
authors.forename: Conrad
authors.initials: C
authors.lastname: Murphy
authors.forename: Vasanthy
authors.initials: V
authors.lastname: Narayanaswami
authors.forename: Robert S
authors.initials: RS
authors.lastname: Kiss
authors.forename: Paul
authors.initials: P
authors.lastname: Hauser
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Guigard
authors.forename: Cyril M
authors.initials: CM
authors.lastname: Kay
authors.forename: Robert O
authors.initials: RO
authors.lastname: Ryan
mesh_terms: D053318:Apolipoprotein E3; D001057:Apolipoproteins E / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D005786:Gene Expression Regulation; D006801:Humans; D008055:Lipids / Q000737:chemistry* / Q000502:physiology*; D010715:Phosphatidylglycerols / Q000737:chemistry; D011485:Protein Binding / Q000502:physiology; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary / Q000502:physiology; D011973:Receptors, LDL / Q000737:chemistry / Q000502:physiology; D012995:Solubility; D013997:Time Factors
chemical_list: D053318:Apolipoprotein E3; D001057:Apolipoproteins E; D005456:Fluorescent Dyes; D008055:Lipids; D010715:Phosphatidylglycerols; D011973:Receptors, LDL; C002773:dimyristoylphosphatidylglycerol