Faculty Collaboration Database

Lipid-triggered conformational switch of apolipophorin III helix bundle to an extended helix organization. J Mol Biol 2002 Aug 09;321(2):201-14

Date

07/30/2002

Pubmed ID

12144779

DOI

10.1016/s0022-2836(02)00618-6

Scopus ID

2-s2.0-0036384368 (requires institutional sign-in at Scopus site)   71 Citations

Abstract

Apolipophorin III (ApoLp-III) from the Sphinx moth, Manduca sexta, is an 18kDa protein that binds reversibly to hydrophobic surfaces generated on metabolizing lipoprotein particles. It is comprised of amphipathic alpha-helices (H1-H5) organized in an up-and-down topology forming a helix bundle in the lipid-free state. Upon interaction with lipids, apoLp-III has been proposed to undergo a dramatic conformational change, involving helix bundle opening about putative hinge loops such that H1, H2 and H5 move away from H3 and H4. In the present study, we examine the relative spatial disposition of H1 and H5 on discoidal phospholipid complexes and spherical lipoproteins. Cysteine residues were engineered at position 8 in H1 and/or at position 138 in H5 in apoLp-III (which otherwise lacks Cys) yielding A8C-, A138C- and A8C/A138C-apoLp-III. Tethering of H1 and H5 by a disulfide bond between A8C and A138C abolished the ability of apoLp-III to transform phospholipid vesicles to discoidal particles, or to interact with lipoproteins, demonstrating that these helices are required to reposition during lipid interaction. Site-specific labeling of A8C/A138C-apoLp-III with N-(1-pyrene)maleimide in the lipid-free state resulted in intramolecular pyrene "excimer" fluorescence emission indicative of spatial proximity between these sites. Upon association with dimyristoylphosphatidylcholine (DMPC) discoidal complexes, the intramolecular excimer was replaced by intermolecular excimer fluorescence due to proximity between pyrene moieties on A8C and A138C in neighboring apoLp-III molecules on the discoidal particle. No excimer emission was observed in the case of pyrene-A8C-apoLp-III/DMPC or pyrene-A138C-apoLp-III/DMPC complexes. However, equimolar mixing of the two labeled single-cysteine mutants prior to disc formation resulted in excimer emission. In addition, intramolecular pyrene excimer formation was diminished upon binding of pyrene-A8C/A138C-apoLp-III to spherical lipoproteins. The data are consistent with repositioning of H1 away from H5 upon encountering a lipid surface, resulting in an extended conformation of apoLp-III that circumscribes the discoidal bilayer particle.

Author List

Sahoo D, Weers PM, Ryan RO, Narayanaswami V

Author

Daisy Sahoo PhD Dean, Professor in the Medicine department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Animals
Apolipoproteins
Cysteine
Dimyristoylphosphatidylcholine
Disulfides
Insect Proteins
Lipid Bilayers
Lipid Metabolism
Lipids
Lipoproteins
Liposomes
Manduca
Mutation
Protein Binding
Protein Conformation
Pyrenes
Spectrometry, Fluorescence