STRUCTURAL STUDIES OF A PEPTIDE ACTIVATOR OF HUMAN LECITHIN-CHOLESTEROL ACYLTRANSFERASE

The synthetic lipid-associating peptide, LAP-20 (VSSLLSSLKEYWSSLKESFS) , activates lecithin-cholesterol acyltransferase (LCAT) despite its la ck of sequence homology to apolipoprotein A-I, the primary in vivo act ivator of LCAT. Using SDS and dodecylphosphocholine (DPC) to model the lipoprotein environment, the structural features responsible for LAP- 20's ability to activate LCAT were studied by optical and two-dimensio nal H-1 NMR spectroscopy. A large blue shift in the intrinsic fluoresc ence of LAP-20 with the addition of detergent suggested that the pepti de formed a complex with the micelles, Analysis of the CD data shows t hat LAP-20 lacks well defined structure in aqueous solution but adopts helical, ordered conformations upon the addition of SDS or DPC. The h elical nature of the peptides in the presence of both lipids was confi rmed by upfield H-alpha NMR secondary shifts relative to random coil v alues. Average structures for both peptides in aqueous solutions conta ining SDS and DPC were generated using distance geometry methods from 329 (SDS) and 309 (DPC) nuclear Overhauser effect-based distance restr aints. The backbone (N, C-alpha, C=O) RMSD from the average structure of an ensemble of 17 out of 20 calculated structures was 0.41 +/- 0.15 Angstrom for LAP-20 in SDS and 0.41 +/- 0.12 Angstrom for an ensemble of 20 out of 20 calculated structures for LAP-20 in DPC. In the prese nce of SDS, the distance geometry and simulated annealing calculations show that LAP-20 adopts a well defined class A amphipathic helix with distinct hydrophobic and hydrophilic faces. A similar structure was o btained for LAP-20 in the presence of DPC, suggesting that both deterg ents may be used interchangeably to model the lipoprotein environment. Conformational features of the calculated structures for LAP-20 are d iscussed relative to models for apolipoprotein A-I activation of LCAT.