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

Jong-Min Han, Tae-Sook Jeong, Woo Song Lee, Inho Choi, and Kyung-Hyun Cho

Korea Res Inst Biosci Biotech, Daejeon

Corresponding Author: chok{at}kribb.re.kr

Val156 of apolipoproteins A-I was found to be a key amino acid in the structure and function of HDL (J. Biol. Chem. (2000) 275: 26821-26827). In order to more precisely determine 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 (T1/2=15±2 min). V156K, however, exhibited faster DMPC clearance activity (T1/2= 6±1 min). In the lipid-bound state, A158E formed a larger reconstituted HDL with palmitoyloleoyl phosphatidylcholine (POPC), around 120 Å, while other mutants and the wild type formed 97 Åof POPC-rHDL. Crosslinking 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 78Å, after 24 hours of incubation in the presence of LDL at 37 oC. V156K and A158E exhibited decreased LCAT activation activity in the POPC-rHDL state, showing less than 2% of wildtype reactivity (apparent Vmax/Km). A158E also displayed markedly different properties in circular dichroism and chymotrypsin digestion analysis, indicating that its three-dimensional structure and its accessibility to proteolytic enzymes are 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.

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