Ca-binding by domain 2 plays a critical role in Gelsolin activation and stabilization

• Gelsolin consists of six homologous domains (G1-G6), each containing a conserved Ca-binding site. Occupation of a subset of these sites enables gelsolin to sever and cap actin filaments in a Ca-dependent manner. Here we present the structures of Ca-free human gelsolin and of Ca-bound human G1-G3 in a complex with actin. These structures closely resemble those previously determined for equine gelsolin. However, the G2 Ca-binding site is occupied in the human G1-G3/actin structure, whereas it is vacant in the equine version. Thermal denaturation studies and actin depolymerization assays indicate that only two Ca-binding events are required for gelsolin activation and filament disassembly. In-depth comparison of the Ca-free and Ca-activated actin-bound human gelsolin structures suggests G2 and G6 to be cooperative in binding Ca2+ and responsible for opening the G2-G6 latch to expose the F-actin binding site on G2. Examination of Ca-binding by G2 in human G1-G3/actin reveals that the Ca2+ locks the G2-G3 interface. Thermal denaturation studies of G2-G3 also indicate that Ca-binding stabilizes this fragment, driving it into the active conformation. The G2 Ca-binding site is mutated in gelsolin from familial amyloidsis (Finnish-type) patients. This disease initially proceeds through protease cleavage of G2, ultimately to produce a fragment that forms amyloid fibrils. The data presented here support a mechanism whereby the loss of Ca-binding by G2 prolongs the lifetime of partially activated intermediate conformations in which the protease cleavage site is exposed.

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