THE HELIX-HINGE-HELIX STRUCTURAL MOTIF IN HUMAN APOLIPOPROTEIN-A-I DETERMINED BY NMR-SPECTROSCOPY

The conformation of a synthetic peptide of 46 residues from apoA-I was investigated by fluorescence, CD, and 2D NMR spectroscopies in lipid- mimetic environments. ApoA-I(142-187) is mainly unstructured in water but helical in SDS or dodecylphosphocholine (DPC), although the peptid e only associates with DPC at approximately the critical micellar conc entration. Solution structures of apoA-I(142-187) were determined by d istance geometry calculations based on 450 (in DPC-d(38)) or 397 (in S DS-d(25)) NOE-derived distance restraints, respectively. Backbone RMSD s for superimposing the two helical regions 146-162 and 168-182 are 0. 98 +/- 0.22 (2.38 +/- 0.20) and 1.99 +/- 0.42 (2.02 +/- 0.21) Angstrom in DPC (SDS), respectively. No interhelical NOE was found, suggesting that helix-helix interactions between the two helical domains in apoA -I(142-187) are unlikely. Similar average, curved helix-hinge-helix st ructures were found in both SDS and DPC micelles with the hydrophobic residues occupying the concave face, indicating that hydrophobic inter actions dominate. Intermolecular NOESY experiments, performed in the p resence of 50% protonated SDS, confirm that the two amphipathic helice s and Y166 in the hinge all interact with the micelle. The involvement of Y166 in lipid binding is supported by fluorescence spectroscopy as well. On the basis of all the data above, we propose a model for the peptide-lipid complexes wherein the curved amphipathic helix-hinge-hel ix structural motif straddles the micelle. The peptide-aided signal as signment achieved for apoA-I(122-187) (66mer) and apoA-I suggests that such a structural motif is retained in the longer peptide and most li kely in the intact protein.