Abstract
Adseverin is a member of the calcium-regulated gelsolin superfamily of actin-binding proteins. Here we report the crystal structure of the calcium-free N-terminal half of adseverin (iA1–A3) and the Ca2+-bound structure of A3, which reveal structural similarities and differences with gelsolin. Solution small-angle X-ray scattering combined with ensemble optimization revealed a dynamic Ca2+-dependent equilibrium between inactive, intermediate and active conformations. Increasing calcium concentrations progressively shift this equilibrium from a main population of inactive conformation to the active form. Molecular dynamics simulations of iA1–A3 provided insights into Ca2+-induced destabilization, implicating a critical role for the A2 type II calcium-binding site and the A2A3 linker in the activation process. Finally, mutations that disrupt the A1/A3 interface increase Ca2+-independent F-actin severing by A1–A3, albeit at a lower efficiency than observed for gelsolin domains G1–G3. Together, these data address the calcium dependency of A1–A3 activity in relation to the calcium-independent activity of G1–G3.
Highlights
Adseverin is a member of the calcium-regulated gelsolin superfamily of actin-binding proteins
Purified recombinant A1–A3, supplemented with 5 mM EGTA, crystallized in space group C2 and its structure was solved by molecular replacement using the structure of G1–G3 excised from inactive full-length gelsolin as the search model and refined to a resolution of 2.9 Å (Fig. 1a and Table 1)
Comparisons with full-length gelsolin show that this interface largely buries the surfaces in inactive forms of A1–A3 (iA1–A3) that bind the C-terminal half of the molecule, suggesting that the crystal environment partially mimics the inactive full-length protein (Supplementary Fig. 1c)
Summary
Adseverin is a member of the calcium-regulated gelsolin superfamily of actin-binding proteins. Crystallographic studies revealed that these binding events involve the association of eight calcium ions, six of which bind at a conserved site on each domain (type II), whereas the remaining two are stabilized by contacts with actin (type I; refs 17–20) Both proteins are able to sequester two monomers of actin, as well as sever and cap actin filaments through distinct Ca2 þ -induced domain rearrangements of the two halves of the molecule[4,17,18,21,22,23]. Small-angle X-ray scattering (SAXS) studies showed a significant calcium-induced increase in the molecular dimensions of gelsolin, and ab initio reconstructions suggested an opening of the two halves and domain rearrangements consistent with the architecture of the active forms observed crystallographically[28]
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