Structural and functional properties of V156K and A158E mutants of apolipoprotein A-I in the lipid-free and lipid-bound states.
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), approximately 120 A, whereas other mutants and the wild type (WT) formed 97 A 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 A, after 24 h of incubation in the presence of low density lipoprotein at 37 degrees C. V156K and A158E exhibited decreased lecithin:cholesterol acyltransferase activation activity in the POPC-rHDL state, showing <2% of WT reactivity (apparent Vmax/Km). 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.
Han
Jong-Min
JM
National Research Laboratory of Lipid Metabolism and Atherosclerosis, Korea Research Institute of Bioscience and Biotechnology, Yuseong-gu, 305-333, South Korea.
Jeong
Tae-Sook
TS
Lee
Woo Song
WS
Choi
Inho
I
Cho
Kyung-Hyun
KH
D016632:Apolipoprotein A-I / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D002918:Chymotrypsin / Q000378:metabolism; D002942:Circular Dichroism; D007700:Kinetics; D008075:Lipoproteins, HDL / Q000378:metabolism*; D010316:Particle Size; D010713:Phosphatidylcholines / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002785:Sterol O-Acyltransferase / Q000378:metabolism
D016632:Apolipoprotein A-I; D008075:Lipoproteins, HDL; D010713:Phosphatidylcholines; D011994:Recombinant Proteins; D002785:Sterol O-Acyltransferase; D002918:Chymotrypsin