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.
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.
Instituto de Investigaciones Bioquimicas de La Plata-Consejo Nacional de Investigaciones Científicas y Técnicas/Universidad Nacional de La Plata, Facultad de Ciencias Médicas, Calles 60 y 120, 1900 La Plata, Argentina.
B
B
Córsico
J D
JD
Toledo
H A
HA
Garda
D000595:Amino Acid Sequence; D016632:Apolipoprotein A-I / Q000097:blood* / Q000737:chemistry* / Q000302:isolation & purification; D001389:Azirines / Q000493:pharmacokinetics; D001665:Binding Sites; D003488:Cyanogen Bromide; D006801:Humans; D019811:Hydroxylamine; D007457:Iodine Radioisotopes; D008051:Lipid Bilayers / Q000737:chemistry*; D008954:Models, Biological; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010713:Phosphatidylcholines / Q000493:pharmacokinetics; D019920:Photoaffinity Labels / Q000493:pharmacokinetics; D017433:Protein Structure, Secondary
D016632:Apolipoprotein A-I; D001389:Azirines; D007457:Iodine Radioisotopes; D008051:Lipid Bilayers; D010446:Peptide Fragments; D010713:Phosphatidylcholines; D019920:Photoaffinity Labels; C088423:1-palmitoyl-2-(decanedioyl mono-(2-iodo-4-(3-trifluoromethyl-3H-diazirin-3-yl)benzyl)ester)glycero-3-phosphocholine; D019811:Hydroxylamine; C033174:3-(trifluoromethyl)-3-(3-iodophenyl)diazirine; D003488:Cyanogen Bromide