Structural and functional properties of V156K and A158E mutants of apolipoprotein A-I in the lipid-free and lipid-bound states

Abstract

Val156 of apolipoprotein A-I (apoA-I) was found to be a key amino acid in the structure and function of high density lipoprotein (HDL) ( J. Biol. Chem. , 275: 26821-26827, 2000). To determine more precisely the functions of the individual amino acids proximal to Val156, serial point mutants of proapoA-I, including V156K, D157K, and A158E, were overexpressed and purified to at least 95% purity. In the lipid-free state, A158E exhibited the most profound self-associative patterns and the least pronounced dimyristoyl phosphatidylcholine (DMPC) clearance activities. In the lipid-bound state, A158E formed a larger reconstituted HDL (rHDL) with palmitoyloleoyl phosphatidylcholine (POPC), ‫ف‬ 120 Å, whereas other mutants and the wild type (WT) formed 97 Å of POPC-rHDL. Cross-linking analysis revealed that A158E-rHDL harbored at least four protein molecules in the particle, while other rHDL conformations contained only two protein molecules. All of the POPC-rHDL produced smaller HDL, around 78 Å, after 24 h of incubation in the presence of low density lipoprotein at 37 ؇ C. V156K and A158E exhibited decreased lecithin:cholesterol acyltransferase activation activity in the POPC-rHDL state, showing Ͻ 2% of WT reactivity (apparent V max / K m ). A158E also displayed markedly different properties in secondary structure, and its accessibility to proteolytic enzymes is different. These results suggest that the two amino acids in helix 6, Val156 and Ala158, are critical to both the structure and function of rHDL. Numerous reports have demonstrated that many of the functions of HDL are highly dependent on the conformation of apoA-I, most notably, the rearrangement of HDL in reverse cholesterol transport (3, 4) and its interaction with the HDL receptor, scavenger receptor class B type I, on the cell surfaces (5, 6). Therefore, a great deal of effort has been focused on the delineation of the structural and functional regions of apoA-I. The central region of apoA-I, spanning residues 143-165, has been implicated in the activation of LCAT and the regulation of high density lipoprotein (HDL) structural rearrangement.Recombinant human proapoA-I has previously been reported to exhibit the same general properties and functions as plasma apoA-I, in both lipid-free and lipid-bound states (7). Previously, Cho and Jonas (8) reported that Valine156 was a key amino acid with regard to the structure and functions of apoA-I, because the recombinant V156E mutant behaved differently from the wild type (WT) in both the lipid-free and lipid-bound states. In the lipid-free state, the V156E mutant exhibited greater protein stability and was quite resistant to self-association, as compared with WT apoA-I. Furthermore, in the palmitoyloleoyl phosphatidylcholine reconstituted HDL (POPCrHDL) state, the V156E did not rearrange its particles to produce smaller particles in the presence of low density lipoprotein (LDL), and exhibited minimal reactivity to LCAT activation. These results indicated that a few of the Abbreviations: ApoA-I, apolipoprot...

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