Conformation of dimeric apolipoprotein A-I Milano on recombinant lipoprotein particles

Apolipoprotein A-I Milano (apoA-IMilano) is a naturally occurring human mutation of wild type apoliprotein A-I (apoA-IWT) having cystine substituted for arginine173. Two molecules of apo-IWT form discs with phospholipid having a defined relationship between the apoA-IWT molecules. ApoA-IMilano forms cystine homodimers that would not allow the protein to adopt the conformation reported for apoA-IWT. The conformational constraints for dimeric apoA-IMilano recombinant high density lipoprotein (rHDL) discs made with phospholipid were deduced from a combination of chemical cross-linking and mass spectrometry. Lysine-selective homo-bifunctional cross-linkers were reacted with homogeneous rHDL having diameters of 78A and 125A. After reduction, cross-linked apoA-IMilano was separated from monomeric apoprotein by gel electrophoresis then subjected to in-gel trypsin digest. Cross-linked peptides were confirmed by MS/MS sequencing. The cross-links provided distance constraints that were used to refine models of lipid-bound dimeric apoA-IMilano. These studies suggest that a single dimeric apoA-IMilano on 78A diameter rHDL girdles the edge of a phospholipid disc assuming a “belt” conformation similar to the “belt” region of apoA-IWT on rHDL. However, the C-terminal end of dimeric apoAIMilano wraps around the periphery of the particle to shield the fatty acid chains from water rather than folding-back onto the “belt” as does apoA-IWT. The two apoA-IMilano dimers on the 125A diameter rHDL encircle the periphery of a phospholipid disc, but appear to reside on the surface of a laminar micelle.