First eight residues of apolipoprotein A-I mediate the C-terminus control of helical bundle unfolding and its lipidation

Abstract

The crystal structure of a C-terminal deletion of apolipoprotein A-I (apoA1) shows a large helical bundle structure in the amino half of the protein, from residues 8 to 115. Using site directed mutagenesis, guanidine or thermal denaturation, cell free liposome clearance, and cellular ABCA1-mediated cholesterol efflux assays, we demonstrate that apoA1 lipidation can occur when the thermodynamic barrier to this bundle unfolding is lowered. The absence of the C-terminus renders the bundle harder to unfold resulting in loss of apoA1 lipidation that can be reversed by point mutations, such as Trp8Ala, and by truncations as short as 8 residues in the amino terminus, both of which facilitate helical bundle unfolding. Locking the bundle via a disulfide bond leads to loss of apoA1 lipidation. We propose a model in which the C-terminus acts on the N-terminus to destabilize this helical bundle. Upon lipid binding to the C-terminus, Trp8 is displaced from its interaction with Phe57, Arg61, Leu64, Val67, Phe71, and Trp72 to destabilize the bundle. However, when the C-terminus is deleted, Trp8 cannot be displaced, the bundle cannot unfold, and apoA1 cannot be lipidated.

Isoform‐specific modification of apolipoprotein E by 4‐hydroxynonenal: protective role of apolipoprotein E3 against oxidative species

“…In control reactions, the change in absorbance of DMPC MLVs in the absence of added protein was followed; in addition, the change in absorbance as a result of adding apoAI, another apolipoprotein known for its ability to rapidly solubilize DMPC vesicles, was monitored as a positive control. For apoAI, the clearance occurred very rapidly with T ½ of ~ 3 min (Table 2); as shown previously by us and other groups [43–45]. The T ½ for unmodified apoE3 was 50.1 ± 3.7 min (Fig.…”

Conformational flexibility of apolipoprotein A-I amino- and carboxy-termini is necessary for lipid binding but not cholesterol efflux

“…However, a slight reduction in efflux occurs with the N-terminal deletions as well (21). Brubaker et al recently demonstrated that limiting the unfolding of the APOA1 N-terminal bundle via an intramolecular disulfide linkage inhibits its lipidation (22).…”

Protein Corona Attenuates the Targeting of Antitumor Sialyl Lewis X-Decorated Liposomes to Vascular Endothelial Cells under Flow Conditions

“…Binding to lipoproteins is thermodynamically preferable for ApoA1, but it also circulates as a free protein in plasma. ApoA1 level in plasma is similar to that of C3 and is about 1–1.5 mg/mL, which makes it one of the most abundant plasma proteins [ 94 ].…”