Abstract
Apolipoprotein A-I (apoA-I) is the main protein of high-density lipoprotein and is comprised of a helical bundle domain and a C-terminal (CT) domain encompassing the last ~65 amino acid residues of the 243-residue protein. The CT domain contains three putative helices (helix 8, 9, and 10) and is critical for initiating lipid binding and harbors sites that mediate self-association of the lipid-free protein. Three lysine residues reside in helix-8 (K195, 206, 208), and three in helix-10 (K226, 238, 239). To determine the role of each CT lysine residue in apoA-I self-association, single, double and triple lysine to glutamine mutants were engineered via site-directed mutagenesis. Circular dichroism and chemical denaturation analysis revealed all mutants retained their structural integrity. Chemical crosslinking and size-exclusion chromatography showed a small effect on self-association when helix-8 lysine residues were changed into glutamine. In contrast, mutation of the three helix-10 lysine residues resulted in a predominantly monomeric protein and K226 was identified as a critical residue. When helix-10 glutamate residues 223, 234, or 235 were substituted with glutamine, reduced self-association was observed similar to that of the helix-10 lysine variants, suggesting ionic interactions between these residues. Thus, helix-10 is a critical part of apoA-I mediating self-association, and disruption of ionic interactions changes apoA-I from an oligomeric state into a monomer. Since the helix-10 triple mutant solubilized phospholipid vesicles at higher rates compared to wild-type apoA-I, this indicates monomeric apoA-I is more potent in lipid binding, presumably because helix-10 is fully accessible to interact with lipids.