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

Abstract

Apolipoprotein A-IV (apoA-IV) is a 376-amino acid exchangeable apolipoprotein made in the small intestine of humans. Although it has many proposed roles in vascular disease, satiety, and chylomicron metabolism, there is no known structural basis for these functions. The ability to associate with lipids may be a key step in apoA-IV functionality. We recently identified a single amino acid, Phe 334 , which seems to inhibit the lipid binding capability of apoA-IV. We also found that an intact N terminus was necessary for increased lipid binding of Phe 334 mutants. Here, we identify Trp 12 and Phe 15 as the N-terminal amino acids required for the fast lipid binding seen with the F334A mutant. Furthermore, we found that individual disruption of putative amphipathic ␣-helices 3-11 had little effect on lipid binding, suggesting that the N terminus of apoA-IV may be the operational site for initial lipid binding. We also provide three independent pieces of experimental evidence supporting a direct intramolecular interaction between sequences near amino acids 12/15 and 334. This interaction could represent a unique "switch" mechanism by which apoA-IV changes lipid avidity in vivo.

Small-angle X-ray Scattering of Apolipoprotein A-IV Reveals the Importance of Its Termini for Structural Stability

“…This has been suggested to be a result of an intact clasp, which is highly dependent on interaction of Phe-334 with the N terminus (Fig. 5E) (19). Consistent with previously published results (19), we observed a similar slow lipid binding profile in the DMPC clearance assay when using only the isolated dimer compared with the mixture of monomers and dimers (Fig.…”

“…Previous work from our laboratory strongly suggests that an interaction between the N and C termini of apoA-IV holds the protein in a conformation that binds lipids poorly (17)(18)(19). Disruption of this "clasp" by introducing the point mutation F334A significantly increased the lipid affinity of apoA-IV (19). Unfortunately, our structure of N/C-terminally truncated apoA-IV did not provide high-resolution structural details pertaining to the clasp mechanism.…”

“…Previous biochemical experiments suggested that interaction of the apoA-IV N and C termini hinders lipid binding (19). Breaking this configuration through site-directed mutagenesis increased the ability of apoA-IV to emulsify lipids.…”

“…However, it is a relatively poor lipid binder (17). Previous work from our laboratory strongly suggests that an interaction between the N and C termini of apoA-IV holds the protein in a conformation that binds lipids poorly (17)(18)(19). Disruption of this "clasp" by introducing the point mutation F334A significantly increased the lipid affinity of apoA-IV (19).…”

ApoA-IV promotes the biogenesis of apoA-IV-containing HDL particles with the participation of ABCA1 and LCAT

“…Furthermore, apoA-IV was reported to have anti-oxidant ( 28 ) and anti-infl ammatory ( 16,29 ) properties, and similarly to apoA-I ( 30 ), and apoE ( 31 ), may also play some role in the development of Alzheimer's disease ( 32 ). A difference between apoA-IV and apoA-I or apoE exists on the contribution of the C-terminal domain of these proteins to the solubilization of dimyristoyl-L -␣ -phosphatidyl-choline (DMPC) phospholipids ( 33,34 ). In the case of apoA-I and apoE, deletion of the C-terminal domain drastically reduced the ability of the truncated forms to solubilize DMPC phospholipids and to associate with preformed HDL ( 35,36 ).…”