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 lack of sequence homology to apolipoprotein A-I, the primary in vivo activator 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-dimensional 1H NMR spectroscopy. A large blue shift in the intrinsic fluorescence of LAP-20 with the addition of detergent suggested that the peptide formed a complex with the micelles. Analysis of the CD data shows that LAP-20 lacks well defined structure in aqueous solution but adopts helical, ordered conformations upon the addition of SDS or DPC. The helical nature of the peptides in the presence of both lipids was confirmed by upfield H alpha NMR secondary shifts relative to random coil values. Average structures for both peptides in aqueous solutions containing SDS and DPC were generated using distance geometry methods from 329 (SDS) and 309 (DPC) nuclear Overhauser effect-based distance restraints. The backbone (N, Calpha, 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 A for an ensemble of 20 out of 20 calculated structures for LAP-20 in DPC. In the presence 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 obtained for LAP-20 in the presence of DPC, suggesting that both detergents may be used interchangeably to model the lipoprotein environment. Conformational features of the calculated structures for LAP-20 are discussed relative to models for apolipoprotein A-I activation of LCAT.
Buchko
G W
GW
Department of Chemistry and Institute of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada V5A 1S6.
Treleaven
W D
WD
Dunne
S J
SJ
Tracey
A S
AS
Cushley
R J
RJ
D000595:Amino Acid Sequence; D016632:Apolipoprotein A-I / Q000737:chemistry / Q000494:pharmacology; D002942:Circular Dichroism; D004789:Enzyme Activation; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D009682:Magnetic Resonance Spectroscopy; D008961:Models, Structural; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000494:pharmacology*; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000378:metabolism*; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
D016632:Apolipoprotein A-I; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; C043948:lipid-associating peptides; D007862:Phosphatidylcholine-Sterol O-Acyltransferase