In the disease
familial amyloidosis, Finnish type (FAF), also known as AGel
amyloidosis (AGel), the mechanism by which point mutations in the
calcium-regulated actin-severing
protein gelsolin lead to
furin cleavage is not understood in the intact
protein. Here, we provide a structural and biochemical characterization of the FAF variants. X-ray crystallography structures of the FAF mutant gelsolins demonstrate that the mutations do not significantly disrupt the
calcium-free conformations of
gelsolin. Small-angle X-ray-scattering (SAXS) studies indicate that the FAF
calcium-binding site mutants are slower to activate, whereas G167R is as efficient as the wild type. Actin-regulating studies of the gelsolins at the
furin cleavage pH (6.5) show that the mutant gelsolins are functional, suggesting that they also adopt relatively normal active conformations. Deletion of
gelsolin domains leads to sensitization to
furin cleavage, and nanobody-binding protects against
furin cleavage. These data indicate instability in the second domain of
gelsolin (G2), since loss or gain of G2-stabilizing interactions impacts the efficiency of cleavage by
furin. To demonstrate this principle, we engineered non-FAF mutations in G3 that disrupt the G2-G3 interface in the
calcium-activated structure. These mutants led to increased
furin cleavage. We carried out molecular dynamics (MD) simulations on the FAF and non-FAF mutant G2-G3 fragments of
gelsolin. All mutants showed an increase in the distance between the center of masses of the 2 domains (G2 and G3). Since G3 covers the
furin cleavage site on G2 in
calcium-activated
gelsolin, this suggests that destabilization of this interface is a critical step in cleavage.