The role of apolipoprotein AI domains in lipid binding.

Abstract

Apolipoprotein AI (apoAI) is the principal protein constituent of high density lipoproteins and it plays a key role in human cholesterol homeostasis; however, the structure of apoAI is not clearly understood. To test the hypothesis that apoAI is organized into domains, three deletion mutants of human apo AI expressed in Escherichia coli were studied in solution and in reconstituted high density lipoprotein particles. Each mutant lacked one of three specific regions that together encompass almost the entire 243 aa sequence of native apoAI (apoAI delta 44-126, apoAI delta 139-170, and apoAI delta 190-243). Circular dichroism spectroscopy showed that the alpha-helical content of lipid-free apoAI delta 44-126 was 27% while the other mutants and native apoAI averaged 55 +/- 2%, suggesting that the missing N-terminal portion contains most of the alpha-helical structure of lipid-free apoAI. ApoAI delta 44-126 exhibited the largest increase in alpha-helix upon lipid binding (125% increase versus an average of 25% for the others), confirming the importance of the C-terminal half of apoAI in lipid binding. Denaturation studies showed that the N-terminal half of apoAI is primarily responsible for alpha-helix stability in the lipid-free state, whereas the C terminus is required for alpha-helix stability when lipid-bound. We conclude that the N-terminal half (aa 44-126) of apoAI is responsible for most of the alpha-helical structure and the marginal stability of lipid-free apoAI while the C terminus (aa 139-243) is less organized. The increase in alpha-helical content observed when native apoAI binds lipid results from the formation of alpha-helix primarily in the C-terminal half of the molecule.