Structure and Stability of Apolipoprotein A-I in Solution and in Discoidal High Density Lipoprotein Probed by Double Charge Ablation and Deletion Mutation

摘要

To identify residues and segments in the central region of apolipoprotein A-I (apoA-I) that are important for the protein structure and stability, we studied the effects of four double charge ablations, D102A/D103A, E110A/E111A, R116V/K118A, and R160V/H162A, and two deletion mutations, Δ(61-78) and Δ(121-142), on the conformation and stability of apoA-I in the lipid-free state and in reconstituted discoidal phospholipid:cholesterol:apoA-I particles (rHDL). The findings suggest that D102/D103 and E110/E111 located in helix 4, and segment(s) between residues 61 and 78 are involved in maintenance of the conformation and stability of apoA-I in both the lipid-free state and in rHDL. R116/K118 located in helix 4 are essential for the conformation and stabilization of apoA-I in rHDL, but not vital for the lipid-free state of the protein. The R160V/H162A substitutions in helix 6 lead to a less compact tertiary structure of lipid-free apoA-I without notable effects on the lipid-free or lipid-bound secondary conformation suggesting involvement of R160/H162 in important inter-helical interactions. The results on the Δ(121-142) mutant, together with our earlier findings, suggest disordered structure of a major segment between residues 121 and 143, likely including residues 131-143, in lipid-free apoA-I. Our findings provide the first experimental evidence for stabilization of rHDL by electrostatic inter-helical interactions, in agreement with the double belt model. The effects of alterations in the conformation and stability of the apoA-I mutants on in vitro and in vivo functions of apoA-I and lipid homeostasis are discussed.