Ca ²⁺ binding by domain 2 plays a critical role in the activation and stabilization of gelsolin

Abstract

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 determined previously 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. In-depth comparison of the Ca-free and Ca-activated, actin-bound human gelsolin structures suggests G2 and G6 to be cooperative in binding Ca 2؉ and responsible for opening the G2-G6 latch to expose the F-actin-binding site on G2. Mutational analysis of the G2 and G6 Ca-binding sites demonstrates their interdependence in maintaining the compact structure in the absence of calcium. Examination of Ca binding by G2 in human G1-G3/actin reveals that the Ca 2؉ locks the G2-G3 interface. Thermal denaturation studies of G2-G3 indicate that Ca binding stabilizes this fragment, driving it into the active conformation. The G2 Ca-binding site is mutated in gelsolin from familial amyloidosis (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.actin ͉ calcium activated ͉ calcium dependent ͉ TIRF

Global Shapes of F-actin Depolymerization-competent Minimal Gelsolins

“…This ␤-sheet edge thus gets exposed and forms an interface with the G3 domain. In addition, Asp-259 (in g2-g3 linker) has been shown to initiate a helix formation in coordination with Thr-260 and condense the otherwise disordered linker to allow close packing of G2 and G3 (7,48). Interestingly, an Asp-260 and Thr-261 are also present at similar positions in this linker region of severin, but the smaller length of this linker or the other specific residues like proline might result in the calcium sensitivity of severin.…”

ArabidopsisVILLIN1 and VILLIN3 Have Overlapping and Distinct Activities in Actin Bundle Formation and Turnover

“…The clear imaging of breaks permits quantitative analyses of severing as well. The technique has been applied to examine the biochemical activity of proteins like plasma gelsolin (Nag et al, 2009) and budding yeast twinfilin (Moseley et al, 2006). In a landmark study, Andrianantoandro and Pollard (2006) demonstrated direct severing by cofilins from fission yeast, Acanthamoeba, and human.…”

Visual Insight into How Low pH Alone Can Induce Actin-severing Ability in Gelsolin under Calcium-free Conditions

“…This might be due to the immense difficulty posed by researchers in attaining diffraction quality crystals of gelsolin at different pH conditions. Interestingly, the two crystal structures of the Ca 2ϩ -activated actinbound N-terminal G1-G3 half of gelsolin were obtained in buffers containing pH 4.7 (29,42). The Ca 2ϩ ion at its binding site in the G2 domain is now resolved in the human gelsolin-actin complex (42), but it could not be resolved in the equine gelsolin (29), which led to questions about how the activation/complexation occurred.…”