The structure of N184K amyloidogenic variant of gelsolin highlights the role of the H-bond network for protein stability and aggregation properties

Abstract

Mutations in the gelsolin protein are responsible for a rare conformational disease known as AGel amyloidosis. Four of these mutations are hosted by the second domain of the protein (G2): D187N/Y, G167R and N184K. The impact of the latter has been so far evaluated only by studies on the isolated G2. Here we report the characterization of full-length gelsolin carrying the N184K mutation and compare the findings with those obtained on the wild type and the other variants.The crystallographic structure of the N184K variant in the Ca 2+ -free conformation shows remarkable similarities with the wild type protein. Only minimal local rearrangements can be observed and the mutant is as efficient as the wild type in severing filamentous actin. However, thermal stability of the pathological variant is compromised in the Ca 2+ -free conditions. These data suggest that the N to K substitution causes a local disruption of the H-bond network in the 2 core of the G2 domain. Such a subtle rearrangement of the connections does not lead to significant conformational changes but severely affects the stability of the protein.

A novel hotspot of gelsolin instability triggers an alternative mechanism of amyloid aggregation

“…its ability to bind and sever actin filaments in a pyrene-labeled fluorometric assay. Under Ca 2+ -free conditions, where GSN adopts the closed and compact conformation, actin binding and severing activity were negligible (0.62 ± 0.01 h −1 for WT) and no pathological mutant analysed so far behaved differently [4] , [28] . The same was true for the A551P and M517R variants (respectively 1.10 ± 0.01 and 0.78 ± 0.01 h −1 ) but not for E553K mutant which surprisingly showed a six times higher severing activity (3.80 ± 0.01 h −1 ) even in the absence of Ca 2+ ( Fig.…”

“…The mechanism of G167R aggregation instead still needs to be fully elucidated. Although this variant shows local destabilization and susceptibility to furin proteolysis similar to the others, this mutation also promotes domain-swapped oligomerization of the protein [27] , [28] , [29] .…”

“…All the amyloidogenic GSN variants characterized so far in domain G2 had increased susceptibility to furin-dependent proteolysis in vitro [24] , [27] , [28] , [38] . To investigate whether A551P, E553K and M517R substitutions affect the proteolysis, we first examined their sensitivity to furin, using the WT and D187N GSN as controls.…”

“…Although irreversible, thermal denaturation of GSN was successfully used to characterize other G2-linked pathological variants (D187N, N184K and G167R) [27] , [28] , [29] . In the absence of Ca 2+ , WT GSN displays prototypical single transition behavior, consistent with the compact and nearly globular conformation of the protein ( Fig.…”

“…2 A). In the presence of the ion, WT GSN unwinds, many inter-domain contacts are modified or lost, and denaturation takes place through a multi-state process [27] , [28] . Surprisingly, all the variants also showed different qualitative behaviors, suggesting a complex role of the mutated residues.…”