The roles of C-terminal helices of human apolipoprotein A-I in formation of high-density lipoprotein particles

Abstract

Apolipoprotein A-I (apoA-I) accepts cholesterol and phospholipids from ATP-binding cassette transporter Al (ABCA1)-expressing cells to form high-density lipoprotein (HDL). Human apoA-I has two tertiary structural domains and the C-terminal domain (approximately amino acids 190–243) plays a key role in lipid binding. Although the high lipid affinity region of the C-terminal domain of apoA-I (residues 223–243) is essential for the HDL formation, the function of low lipid affinity region (residues 191–220) remains unclear. To evaluate the role of residues 191–220, we analyzed the structure, lipid binding properties, and HDL formation activity of Δ191–220 apoA-I, in comparison to wild-type and Δ223–243 apoA-I. Although deletion of residues 191–220 has a slight effect on the tertiary structure of apoA-I, the Δl91–220 variant showed intermediate behavior between wild-type and Δ223–243 regarding the formation of hydrophobic sites and lipid interaction through the C-terminal domain. Physicochemical analysis demonstrated that defective lipid binding of Δl91–220 apoA-I is due to the decreased ability to form α-helix structure which provides the energetic source for lipid binding. In addition, the ability to form HDL particles in vitro and induce cholesterol efflux from ABC Al-expressing cells of Δ191–220 apoA-I was also intermediate between wild-type and Δ223–243 apoA-I. These results suggest that despite possessing low lipid affinity, residues 191–220 play a role in enhancing the ability of apoA-I to bind to and solubilize lipids by forming α-helix upon lipid interaction. Our results demonstrate that the combination of low lipid affinity region and high lipid affinity region of apoA-I is required for efficient ABCA1-dependent HDL formation.

Direct detection of ABCA1-dependent HDL formation based on lipidation-induced hydrophobicity change in apoA-I

“…Furthermore, the apoA-I-POLARIC concentration dependence of cholesterol effl ux ( K m , 1.8 ± 0.6 g/ml) was consistent with the reported property of wild-type apoA-I ( Fig. 2B ) ( 7,28 ). Therefore, labeling with POLARIC-maleimide affected neither the structure nor the function of apoA-I, and apoA-I-POLARIC possessed the intact ABCA1-dependent HDL formation activity.…”

“…Concentrated medium was fractionated by gel fi ltration chromatography on a Superdex 200 column calibrated by the proteins of known diameter (particle diameter range, 6.1-17.0 nm), and 1.25 ml fractions were collected ( 28 ). Fluorescence intensity and the amount of cholesterol in each fraction were analyzed as described above and normalized using BSA.…”

Probing the C-terminal domain of lipid-free apoA-I demonstrates the vital role of the H10B sequence repeat in HDL formation

“…Thus, the helical conformation in this region may result in interaction of H10B with the N-terminal helix bundle. Previous research has also suggested that the C-terminal helix is located near the N-terminal helix bundle and there is interaction between them (25,26,45). Due to the increase in the unfolding cooperativity, the stability of the elongation apoA-I variants is increased or remains similar compared with WT apoA-I (Fig.…”

“…The entire C-terminal domain converts into helical structure upon lipid binding (14). Residues 191-220 play a role in enhancing the ability of apoA-I to bind to and solubilize lipids by forming helix upon lipid interaction (45). All of these studies suggest that the C-terminal domain has a dynamic structure and plays an important role during the lipid-binding process by shifting from disordered into helical structure.…”

“…The detergent has been used in numerous studies to investigate the effects of lipid environment in the helical WT apoA-I (45). Considering that the 191-220 segment may form a loop, this deletion might shift the H10B helix interaction with the N-terminal helix bundle causing the destabilization, while the elongation apoA-I variants remain similar or have increased stability.…”