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 invo lved in this process, we examined residue conservation and topology of apoA -I from all known species. We observed that the hydrophobic/hydrophilic int erface of helix 143-164 contains a cluster of three strictly conserved argi nine residues (R149, R153, and R160), and that these residues create the on ly significant positive electrostatic potential around apoA-I. To test the importance of R149, R153, and R160 in LCAT activation, we generated a serie s of mutant proteins. These had fluorescence emission, secondary structure, and lipid-binding properties comparable to those of wild-type apoA-I. Muta tion of conserved residues R149, R153, and R160 drastically decreased LCAT activity on lipid-protein complexes, whereas control mutations (E146Q, D150 N, D157N, R171Q, and A175R) did not decrease LCAT activity by more than 55% . The markedly decreased activities of mutants R149, R153, and R160 resulte d from a decrease in the maximal reaction velocity V-max because the appare nt Michaelis-Menten constant K-m values were similar for the mutant and wil d-type apoA-I proteins. These data suggest that R149, R153, and R160 partic ipate in apoA-I-mediated activation of LCAT, and support the "belt" model f or discoidal rHDL. In this model, residues R149, R153, and R160 do not form salt bridges with the antiparallel apoA-I monomer, but instead are pointin g toward the surface of the disc, enabling interactions with LCAT.