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J. Biol. Chem., Vol. 281, Issue 51, 39294-39299, December 22, 2006

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Lipid-induced Extension of Apolipoprotein E Helix 4 Correlates with Low Density Lipoprotein Receptor Binding Ability^*

Vinita Gupta, Vasanthy Narayanaswami, Madhu S. Budamagunta, Taichi Yamamato, John C. Voss, and Robert O. Ryan¹

From the Center for the Prevention of Obesity, Cardiovascular Disease and Diabetes, Children's Hospital Oakland Research Institute, Oakland, California 94609 and Department of Biochemistry and Molecular Medicine, School of Medicine, University of California, Davis, California 95616

Apolipoprotein E (apoE) serves as a ligand for the low density lipoprotein receptor (LDLR) only when bound to lipid. The N-terminal domain of lipid-free apoE exists as globular 4-helix bundle that is conferred with LDLR recognition ability after undergoing a lipid binding-induced conformational change. To investigate the structural basis for this phenomenon, site-directed spin label electron paramagnetic resonance spectroscopy experiments were conducted, focusing on the region near the C-terminal end of helix 4 (Ala-164). Using C112S apoE-N-terminal as template, a series of single cysteine substitution variants (at sequence positions 161, 165, 169, 173, 176, and 181) were produced, isolated, and labeled with the nitroxide probe, methane thiosulfonate. Electron paramagnetic resonance analysis revealed that lipid association induced fixed secondary structure in a region of the molecule known to exist as random coil in the lipid-free state. In a complementary approach, site-directed fluorescence analysis using an environmentally sensitive probe indicated that the lipid-induced transition of this region of the protein to helix was accompanied by relocation to a more hydrophobic environment. In studies with full-length apoE single Cys variants, a similar random coil to stable backbone transition was observed, consistent with the concept that lipid interaction induced an extension of helix 4 beyond the boundary defining its lipid-free conformation. This structural transition likely represents a key conformational change necessary for manifestation of the LDLR recognition properties of apoE.

Received for publication, August 23, 2006 , and in revised form, October 31, 2006.

^* This work was supported by the National Institutes of Health Grant (HL-64159). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Center for the Prevention of Obesity, Cardiovascular Disease and Diabetes, Children's Hospital Oakland Research Inst., 5700 Martin Luther King Jr. Way, Oakland, CA 94609. Tel.: 510-450-7645; Fax: 510-450-7910; E-mail: rryan{at}chori.org.

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