Evidence for a Central Apolipoprotein A-I Domain Loosely Bound to Lipids in Discoidal Lipoproteins That Is Capable of Penetrating the Bilayer of Phospholipid Vesicles

Betina Córsico,Juan Domingo Toledo,Horacio Alberto Garda

doi:10.1074/jbc.m011533200

Abstract

Previous evidence indicated that discoidal reconstituted high density lipoproteins (rHDL) of apolipoprotein A-I (apoA-I) can interact with lipid membranes (Tricerri, M. A., Córsico, B., Toledo, J. D., Garda, H. A., and Brenner, R. R. (1998) Biochim. Biophys. Acta 1391, 67-78). With the aim of studying this interaction, photoactivable reagents and protein cleavage with CNBr and hydroxylamine were used. The generic hydrophobic reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine gave information on the apoA-I regions in contact with the lipid phase in the rHDL discs. Two protein regions loosely bound to lipids were detected: a C-terminal domain and a central one located between residues 87 and 112. They consist of class Y amphipathic alpha-helices that have a different distribution of the charged residues in their polar faces by comparison with class A helices, which predominate in the rest of the apoA-I molecule. The phospholipid analog 1-O-hexadecanoyl-2-O-[9-[[[2-[125I]iodo-4-(trifluoro-methyl-3-H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine, which does not undergo significant exchange between membranes and lipoproteins, was used to identify the apoA-I domain directly involved in the interaction of rHDL discs with membranes. By incubating either rHDL or lipid-free apoA-I with lipid vesicles containing 125I-TID-PC, only the 87-112 apoA-I segment becomes labeled after photoactivation. These results indicate that the central domain formed by two type Y helices swings away from lipid contact in the discoidal lipoproteins and is able to insert into membrane bilayers, a process that may be of great importance for the mechanism of cholesterol exchange between high density lipoproteins and cell membranes.

Highlights

A high conformational flexibility in apolipoprotein A-I (apoA-I) is needed for its existence in different states: lipid-free, lipid-poor, and discoidal or spherical lipoproteins of different size
These results indicate that the central domain formed by two type Y helices swings away from lipid contact in the discoidal lipoproteins and is able to insert into membrane bilayers, a process that may be of great importance for the mechanism of cholesterol exchange between high density lipoproteins and cell membranes
In the spherical ␣-HDL, it has been proposed [25] that the amphipathic helices are oriented parallel to the surface of the phospholipid monolayer with the hydrophobic faces embedded into the hydrocarbon region and with the hydrophilic faces interacting with the phospholipid vesicles; rHDL, reconstituted high density lipoproteins; PAGE, polyacrylamide gel electrophoresis; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine

Summary

Introduction

A high conformational flexibility in apoA-I is needed for its existence in different states: lipid-free, lipid-poor, and discoidal or spherical lipoproteins of different size. The same apoA-I fragment becomes almost exclusively labeled by the incubation of these 125I-TID-PC-containing vesicles with the different discoidal complexes (Lp2, Lp2C, Lp4, or Lp4C) or even with lipid-free apoA-I, a different level of labeling can be observed, which is possibly due to differences in affinity or the ability to interact as previously reported [49].

Results

Conclusion