Abstract Body: Introduction: MTP is essential for lipoprotein biosynthesis. Crystal structure, mutagenesis and human mutations have shown that A- and C-sheets within the C-terminal lipid binding domain of MTP are critical for function. We found that these sheets are joined with several loops. Loops stabilize protein structure and facilitate ligand recognition and binding. However, the role of connecting loops in MTP remains uncharacterized.
Hypothesis: Loops in the lipid transfer domain are critical for MTP function.
Methods: Amino acids within loop regions were mutated to alanine via site-directed mutagenesis. MTP deficient human hepatoma Huh-7 cells were transfected with plasmids expressing wild type and mutant MTP. After 48-60 h, MTP expression and lipid transfer activity were studied by western blot and MTP activity assays, respectively. ApoB100 levels in the overnight conditioned media were quantified by ELISA. p < 0.05 was considered significant.
Results: We identified two flexible loops in the A-sheet (A-Loop 1 of 769-772 and A-Loop 2 of 819-824 amino acids). In A-Loop 1, W770A mutation abolished lipid transfer activity and apoB secretion. Mutations in the A-Loop 2 had no significant effect on MTP function. Three flexible loops were identified in the C-sheet. Individual mutagenesis of S638, G639, S640 and G641 in the C-Loop 1 resulted in complete loss of lipid transfer activity and apoB secretion. C-Loop 2 (G709-S688) contains two small helices (H1 and H2) and a coiled region. Mutations at L671 and E672 in H1 disrupted MTP function while mutagenesis of other amino acids had no effect. C-loop 3 consists of one helix (H3). Mutagenesis of L713 and L718 in H3 led to loss of MTP lipid transfer activity and apoB secretion.
Conclusions: These studies show that flexible loops within the C-sheet are critical for MTP function. Every amino acid in the SGSG C-loop 1 connecting the β2 and β3-strands of C-sheet is essential for MTP function. It is likely that this motif plays a role in substrate recognition and entry. Helices H1 and H3 are close to the lipid binding site in MTP, and they may be part of the lipid binding cavity. Tryptophan residues play a role in membrane anchoring in other proteins. It is likely that W770 might interact with membranes to facilitate lipid extraction. This information may be useful in designing gain of function and partial MTP inhibition for the treatment of steatosis and hypercholesterolemia.