Specific Sequences in the N and C Termini of Apolipoprotein A-IV Modulate Its Conformation and Lipid Association

Abstract

Apolipoprotein (apoA-IV) is a 376-residue exchangeable apolipoprotein that may play a number of important roles in lipid metabolism, including chylomicron assembly, reverse cholesterol transport, and appetite regulation. In vivo, apoA-IV exists in both lipidpoor and lipid-associated forms, and the balance between these states may determine its function. We examined the structural elements that modulate apoA-IV lipid binding by producing a series of deletion mutants and determining their ability to interact with phospholipid liposomes. We found that the deletion of residues 333-343 strongly increased the lipid association rate versus native apoA-IV. Additional mutagenesis revealed that two phenylalanine residues at positions 334 and 335 mediated this lipid binding inhibitory effect. We also observed that residues 11-20 in the N terminus were required for the enhanced lipid affinity induced by deletion of the C-terminal sequence. We propose a structural model in which these sequences can modulate the conformation and lipid affinity of apoA-IV.

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

“…1 shows that WT apoA-IV was relatively ineffective at clearing DMPC liposomes compared with the ⌬333-343 mutant, as shown previously (19). However, simultaneous deletion of residues 11-20 in the ⌬333-343 background reverted this fast mutant to the slow phenotype of the WT.…”

“…Site-directed Mutagenesis-Deletion and point mutants of human apoA-IV were created by site-directed mutagenesis as described previously (18,19) in the bacterial expression vector, pET30 (Novagen). Complete DNA sequences of positively screening clones were verified by sequencing the entire cDNA.…”

“…We found that the mutation of either phenylalanine 334 or 335 to alanine drastically increased the rate at which apoA-IV is able to reorganize 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) liposomes. However, if amino acids 11-20 were deleted, the mutant reversed to the slow lipid binding of wild type apoA-IV (19). In the current work, we have determined the exact residues present in the N terminus that are responsible for reversion to the slow lipid-binding phenotype.…”

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

“…This implies that an increase in lipid binding might be associated with a more fluid core bundle. A fluid core bundle was also observed in the N-terminal truncation apoA-IV 62-376 , but this form remains a poor lipid binder due to deletion of important lipid binding determinants (18). Taken together, these results fundamentally redefine the clasp mechanism as an interaction between the termini and the main bundle instead of just the termini and provide a structural explanation for how the N and C termini can impact lipid binding by altering the overall bundle architecture.…”

“…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).…”