Effects of the Iowa and Milano Mutations on Apolipoprotein
A‑I Structure and Dynamics Determined by Hydrogen Exchange
and Mass Spectrometry
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Abstract
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