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 apolipoprotein A-I (apoA-IWT) having cystine substituted for arginine173. Two molecules of apo-IWT form disks 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-I Milano recombinant high-density lipoprotein (rHDL) disks made with phospholipid were deduced from a combination of chemical cross-linking and mass spectrometry. Lysine-selective homobifunctional cross-linkers were reacted with homogeneous rHDL having diameters of 78 and 125 Å. After reduction, cross-linked apoA-IMilano was separated from monomeric apoprotein by gel electrophoresis and 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-I Milano. These studies suggest that a single dimeric apoA-I Milano on 78 Å diameter rHDL girdles the edge of a phospholipid disk assuming a ";belt"; conformation similar to the ";belt"; region of apoA-IWT on rHDL. However, the C-terminal end of dimeric apoA-IMilano 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-I Milano dimers on a 125 Å diameter rHDL do not encircle the periphery of a phospholipid disk but appear to reside on the surface of a laminar micelle.