Effects of the Iowa and Milano mutations on apolipoprotein A-I structure and dynamics determined by hydrogen exchange and mass spectrometry.
The Iowa point mutation in apolipoprotein A-I (G26R) leads to a systemic amyloidosis condition, and the Milano mutation (R173C) is associated with hypoalphalipoproteinemia, a reduced plasma level of high-density lipoprotein. To probe the structural effects that lead to these outcomes, we used amide hydrogen-deuterium exchange coupled with a fragment separation/mass spectrometry analysis (HX MS). The Iowa mutation inserts an arginine residue into the nonpolar face of an α-helix that spans residues 7-44 and causes changes in structure and structural dynamics. This helix unfolds, and other helices in the N-terminal helix bundle domain are destabilized. The segment encompassing residues 116-158, largely unstructured in wild-type apolipoprotein A-I, becomes helical. The helix spanning residues 81-115 is destabilized by 2 kcal/mol, increasing the small fraction of time it is transiently unfolded to ≥1%, which allows proteolysis at residue 83 in vivo over time, releasing an amyloid-forming peptide. The Milano mutation situated on the polar face of the helix spanning residues 147-178 destabilizes the helix bundle domain only moderately, but enough to allow cysteine-mediated dimerization that leads to the altered functionality of this variant. These results show how the HX MS approach can provide a powerful means of monitoring, in a nonperturbing way and at close to amino acid resolution, the structural, dynamic, and energetic consequences of biologically interesting point mutations.
Lipid Research Group, Gastroenterology, Hepatology and Nutrition Division, Children's Hospital of Philadelphia, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania 19104-4318, United States.
Palaniappan Sevugan
PS
Chetty
Maki
M
Ohshiro
Hiroyuki
H
Saito
Padmaja
P
Dhanasekaran
Sissel
S
Lund-Katz
Leland
L
Mayne
Walter
W
Englander
Michael C
MC
Phillips
D000595:Amino Acid Sequence; D016632:Apolipoprotein A-I / Q000737:chemistry* / Q000235:genetics*; D041961:Deuterium Exchange Measurement; D006801:Humans; D006859:Hydrogen / Q000737:chemistry; D013058:Mass Spectrometry; D017354:Point Mutation; D017433:Protein Structure, Secondary / Q000187:drug effects
D016632:Apolipoprotein A-I; D006859:Hydrogen