Conformation of dimeric apolipoprotein A-I milano on recombinant lipoprotein particles.
Apolipoprotein A-I Milano (apoA-I(Milano)) is a naturally occurring human mutation of wild-type apolipoprotein A-I (apoA-I(WT)) having cystine substituted for arginine(173). Two molecules of apo-I(WT) form disks with phospholipid having a defined relationship between the apoA-I(WT) molecules. ApoA-I(Milano) forms cystine homodimers that would not allow the protein to adopt the conformation reported for apoA-I(WT). 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 A. After reduction, cross-linked apoA-I(Milano) 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 A diameter rHDL girdles the edge of a phospholipid disk assuming a "belt" conformation similar to the "belt" region of apoA-I(WT) on rHDL. However, the C-terminal end of dimeric apoA-I(Milano) 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-I(WT). The two apoA-I(Milano) dimers on a 125 A diameter rHDL do not encircle the periphery of a phospholipid disk but appear to reside on the surface of a laminar micelle.
Department of Pathology, Center for Lipid Science, Wake Forest University Health Sciences, Medical Center Boulevard, Winston-Salem, North Carolina 27157, USA.
Shaila
S
Bhat
Mary G
MG
Sorci-Thomas
Laura
L
Calabresi
Michael P
MP
Samuel
Michael J
MJ
Thomas
D000595:Amino Acid Sequence; D016632:Apolipoprotein A-I / Q000737:chemistry*; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry
D016632:Apolipoprotein A-I; D011994:Recombinant Proteins; C080631:apolipoprotein A-I Milano