The role of gelsolin domain 3 in familial amyloidosis (Finnish type)

Abstract

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.

Keywords: AGel amyloidosis; FAF; amyloid; gelsolin; structure.

Fig. 1.

Schematic representation of the structure of full-length gelsolin. Figure is colored with domain G1 in red, domain G2 in light green, domain G3 in yellow, domain G4 in purple, domain G5 in dark green, and domain G6 in orange (Center). The black square focuses on the FAF mutation region of domain G2. The 2Fo-Fc electron density map of the residues is shown as a gray grid comparing the 3 different mutants contoured at 1 σ, with (A) Asn187, (B) Native Asp187, (C) Tyr187, (D) Native Gly167, and (E) Arg167. Mutated residues are highlighted with a red star. (F) Comparison of the root-mean-square deviation values of the native gelsolin structure versus mutants.

Fig. 2.

FAF gelsolin activity at pH 6.5. (A) Actin nucleation assay with FAF gelsolin mutants. G-actin was incubated with different gelsolin mutants at a molar ratio of 1:100 for 1 h followed by the addition of 10× KMEI buffer. FAF mutants nucleate actin filaments similarly as wild-type gelsolin. Data represent the average of 3 experiments. (B–E) FAF gelsolins sever F-actin. Sedimentation assays of FAF gelsolin were carried out under EGTA and calcium conditions. F-actin was incubated with different concentrations of wild-type and mutant gelsolins for 1 h at room temperature and subjected to ultracentrifugation, and the supernatants (S) and pellets (P) were analyzed by SDS/PAGE. (B) WT, (C) 187N, (D) 187Y, (E) 167R. Right, 1.5 mM EGTA. Left, 1 mM calcium chloride.

Fig. 3.

Kratky plots of the time points of calcium activation of (A) WT, (B) D187Y, (C) D187N, and (D) G167R gelsolins. In all panels, the individual frames of the stopped-flow experiments, with 4 mg/mL protein in the presence of 1 mM Ca²⁺ are shown color-coded from blue (130 ms) to red (400 ms) for increasing time points. For the WT gelsolin, steady state continuous flow plots are included, EGTA (solid black line) and calcium (dashed black line). For reference, these WT continuous flow profiles are also shown as gray lines in the mutant panels. (E) Time dependence of Rg values. (F and G) Thermal shift assays. Thermal denaturation studies of WT and mutants in the presence of EGTA (F) or calcium chloride (G). Values of the midpoints of the WT thermal denaturation profiles are indicated.

Fig. 4.

Gelsolin FAF mutants are protected by Nb11 from furin cleavage. Susceptibility to furin cleavage was tested in vitro for both full-length (FL) and G2G3 constructs and analyzed by SDS/PAGE. Incubation of the (A) FL mutants and (B) G2G3 mutants with furin for 3 h. Production of cleaved fragments is observed for the mutants, which is more efficient for G2G3 than FL under the same conditions. Inclusion of Nb11 inhibits the furin cleavage in A and B. “Nb” refers to the inclusion of nanobody. Below, the extent of cleavage was assessed by densitometry as the average value from three gels. (C) Furin cleavage was tested for the non-FAF G2G3 mutants, which show a cleaved gel band assessed after 3 h. Inclusion of Nb11 inhibited the appearance of this gel band. Time course and titrations for the furin assay are found in SI Appendix, Figs. S3 and S4. Statistical tests were applied on differences observed in the percentage of cleaved proteins. Data are the average of three independent experiments ±SD. Student’s t test (2-sided) was used for statistical analysis. (D) Cartoons indicate the relative positions of the mutation sites (stars) relative to G2 (green), G3 (yellow), the cleavage site (red cross), calcium-binding sites (black ovals), and the Nb11-binding site.

Fig. 5.

Molecular dynamics simulations of the WT and FAF mutants. (A) RMSD of G2G3 relative to the native structure of the calcium-bound active conformation (PDB 3FFK) as a function of simulation time. (B) Potential of mean force as a function of COM distance. The PMF profiles and the errors were calculated by the averages and SDs of the results from 3 independent umbrella-sampling simulations.