Three arginine residues in apolipoprotein A-I are critical for activation of lecithin:cholesterol acyltransferase.
Previous studies have suggested that the helical repeat formed by residues 143;-164 of apolipoprotein A-I (apoA-I) contributes to lecithin:cholesterol acyltransferase (LCAT) activation. To identify specific polar residues involved in this process, we examined residue conservation and topology of apoA-I from all known species. We observed that the hydrophobic/hydrophilic interface of helix 143;-164 contains a cluster of three strictly conserved arginine residues (R149, R153, and R160), and that these residues create the only significant positive electrostatic potential around apoA-I. To test the importance of R149, R153, and R160 in LCAT activation, we generated a series of mutant proteins. These had fluorescence emission, secondary structure, and lipid-binding properties comparable to those of wild-type apoA-I. Mutation of conserved residues R149, R153, and R160 drastically decreased LCAT activity on lipid-protein complexes, whereas control mutations (E146Q, D150N, D157N, R171Q, and A175R) did not decrease LCAT activity by more than 55%. The markedly decreased activities of mutants R149, R153, and R160 resulted from a decrease in the maximal reaction velocity V(max) because the apparent Michaelis-Menten constant K(m) values were similar for the mutant and wild-type apoA-I proteins. These data suggest that R149, R153, and R160 participate in apoA-I-mediated activation of LCAT, and support the "belt" model for discoidal rHDL. In this model, residues R149, R153, and R160 do not form salt bridges with the antiparallel apoA-I monomer, but instead are pointing toward the surface of the disc, enabling interactions with LCAT. - Roosbeek, S., B. Vanloo, N. Duverger, H. Caster, J. Breyne, I. De Beun, H. Patel, J. Vandekerckhove, C. Shoulders, M. Rosseneu, and F. Peelman. Three arginine residues in apolipoportein A-I are critical for activation of lecithin:cholesterol acyltransferase J. Lipid Res. 2001. 42: 31;-40.
Roosbeek
S
S
Laboratory for Lipoprotein Chemistry, Department of Biochemistry, Ghent University, B-9000 Ghent, Belgium.
Vanloo
B
B
Duverger
N
N
Caster
H
H
Breyne
J
J
De Beun
I
I
Patel
H
H
Vandekerckhove
J
J
Shoulders
C
C
Rosseneu
M
M
Peelman
F
F
D000595:Amino Acid Sequence; D000818:Animals; D016632:Apolipoprotein A-I / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D001120:Arginine / Q000737:chemistry / Q000494:pharmacology*; D017124:Conserved Sequence / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism; D011485:Protein Binding; D016415:Sequence Alignment; D055672:Static Electricity
D016632:Apolipoprotein A-I; D010743:Phospholipids; D001120:Arginine; D007862:Phosphatidylcholine-Sterol O-Acyltransferase