Contributions of the N- and C-Terminal Helical Segments to the Lipid-Free Structure and Lipid Interaction of Apolipoprotein A-I

Abstract

The tertiary structure of lipid-free apolipoprotein (apo) A-I in the monomeric state comprises two domains: a N-terminal alpha-helix bundle and a less organized C-terminal domain. This study examined how the N- and C-terminal segments of apoA-I (residues 1-43 and 223-243), which contain the most hydrophobic regions in the molecule and are located in opposite structural domains, contribute to the lipid-free conformation and lipid interaction. Measurements of circular dichroism in conjunction with tryptophan and 8-anilino-1-naphthalenesulfonic acid fluorescence data demonstrated that single (L230P) or triple (L230P/L233P/Y236P) proline insertions into the C-terminal alpha helix disrupted the organization of the C-terminal domain without affecting the stability of the N-terminal helix bundle. In contrast, proline insertion into the N terminus (Y18P) disrupted the bundle structure in the N-terminal domain, indicating that the alpha-helical segment in this region is part of the helix bundle. Calorimetric and gel-filtration measurements showed that disruption of the C-terminal alpha helix significantly reduced the enthalpy and free energy of binding of apoA-I to lipids, whereas disruption of the N-terminal alpha helix had only a small effect on lipid binding. Significantly, the presence of the Y18P mutation offset the negative effects of disruption/removal of the C-terminal helical domain on lipid binding, suggesting that the alpha helix around Y18 concealed a potential lipid-binding region in the N-terminal domain, which was exposed by the disruption of the helix-bundle structure. When these results are taken together, they indicate that the alpha-helical segment in the N terminus of apoA-I modulates the lipid-free structure and lipid interaction in concert with the C-terminal domain.

Modulation of Apolipoprotein A-IV Lipid Binding by an Interaction between the N and C Termini

“…It is well known that similar helical bundles within apoE and apoA-I open up when structures at the C terminus interact with lipid. This event appears to trigger an extensive conformational change resulting in the sequential binding of each of the helical segments to form an extended lipid-bound conformation and solubilized lipid (39). In apoA-IV, we proposed that the region near residue 334/335 participates in an intramolecular interaction with the N terminus to "lock" the helical bundle in a closed conformation that is unable to rapidly open in the presence of lipid.…”

The Structure of Apolipoprotein A-I in High Density Lipoproteins

“…The thermodynamic drive to minimize the aqueous exposure of these hydrophobic surfaces is probably the major mediator of protein folding, whether these surfaces are present in a lipid-free state in which the nonpolar helical faces sequester within the protein or in the lipidated state where they likely contact aliphatic regions of lipid assemblies. More information on how amphipathic helices mediate apoA-I lipid binding can be found in recent work from the Phillips laboratory (12) and in the excellent review series by Brouillette et al (13).…”

Amyloidogenic Mutation Promotes Fibril Formation of the N-terminal Apolipoprotein A-I on Lipid Membranes

“…Most recently, lipid vesicles were shown to accelerate the nucleation step of ␣-synuclein (35) and amyloid-␤ peptide (36), triggering conversion into the aggregated state. Because apoA-I has a strong ability to bind to lipid membranes (37) and both the N-and C-terminal regions are involved in lipid binding (38), it is expected that membrane binding has a key role in the aggregation pathway of the N-terminal fragment of apoA-I. However, little is known about the effect of the lipid environment on the amyloidogenic property of apoA-I to date (39).…”