Abstract 408: Essential Domain Transition Involving Central Helical Repeats Prevent Lipid-Free ApoA-I from Acquiring Lipid

Abstract

Plasma high density lipoprotein (HDL) concentration is negatively correlated with the occurrence of coronary heart disease in the human population. Because apoA-I is the main protein constituent of HDL, a thorough understanding of apoA-I structural topology is essential for elucidating its ability to package and mobilize cholesterol for catabolism. To determine which of the 10 helical repeats within apoA-I participates in the structural transitions that drive unfolding of the 4-helix bundle, we created several loss of function mutations. Published three-dimensional coordinates of full-length lipid-free apoA-I were used to predict amino acids having spatial separations of 3-5Å within the 4-helix bundle. Based on these predictions, we proposed that specific targeted double cysteine residue substitutions could form disulfide linkages and prevent “opening” of a critical domain required for unfolding of the apoA-I 4-helix bundle when exposed to lipid. To test the importance of helical repeats 4, 5, 6 and 7, double cysteine mutants D103C-R177C apoA-I and F104C-H162C apoA-I were created, expressed and purified using established procedures. Mass spectrometry combined with MS/MS sequencing was used to verify the “locked” disulfide form of each double cysteine substitution mutants. Using 20% SDS-PAGE we show that electrophoretic mobility-shift distinguishes between “locked” or -DTT versus “unlocked” or +DTT form for each of the mutant apoA-I proteins. Particle formation was tested for each mutant by measuring the formation of recombinant HDL (rHDL) using cholate dialysis, as well as, the formation of nascent HDL (nHDL) from ABCA1 expressing cells. Examination of the size of rHDL and nHDL particles formed suggests that unfolding of lipid-free apoA-I to acquire lipid involves the “locked” or restricted helical repeats 4-7. In conclusion, when both double cysteine apoA-I mutants exist in their “locked” conformation evidence of impaired particle formation was observed confirming the existence of the apoA-I 4 helix bundle in lipid free state and the role of central helical repeats 4-7 in lipid binding and particle formation.