  AGel amyloidosis, formerly known as familial amyloidosis of the Finnish-type,
is caused by pathological aggregation of proteolytic fragments of plasma
gelsolin. So far, four mutations in the gelsolin gene have been reported as
responsible for the disease. Although D187N is the first identified variant and
the best characterized, its structure has been hitherto elusive. Exploiting a
recently-developed nanobody targeting gelsolin, we were able to stabilize the
G2 domain of the D187N protein and obtained, for the first time, its
high-resolution crystal structure. In the nanobody-stabilized conformation, the
main effect of the D187N substitution is the impairment of the calcium binding
capability, leading to a destabilization of the C-terminal tail of G2. However,
molecular dynamics simulations show that in the absence of the nanobody,
D187N-mutated G2 further misfolds, ultimately exposing its hydrophobic core and
the furin cleavage site. The nanobody's protective effect is based on the
enhancement of the thermodynamic stability of different G2 mutants (D187N,
G167R and N184K). In particular, the nanobody reduces the flexibility of
dynamic stretches, and most notably decreases the conformational entropy of the
C-terminal tail, otherwise stabilized by the presence of the Ca2+ ion. A
Caenorhabditis elegans-based assay was also applied to quantify the proteotoxic
potential of the mutants and determine whether nanobody stabilization
translates into a biologically relevant effect. Successful protection from G2
toxicity in vivo points to the use of C. elegans as a tool for investigating
the mechanisms underlying AGel amyloidosis and rapidly screen new therapeutics.
