Calcium Ion Exchange in Crystalline Gelsolin

Abstract

Gelsolin is a calcium and pH-sensitive modulator of actin filament length. Here, we use X-ray crystallography to examine the extraction and exchange of calcium ions from their binding sites in different crystalline forms of the activated N and C-terminal halves of gelsolin, G1-G3 and G4-G6, respectively. We demonstrate that the combination of calcium and low pH activating conditions do not induce conformational changes in G4-G6 beyond those elicited by calcium alone. EGTA is able to remove calcium ions bound to the type I and type II metal ion-binding sites in G4-G6. Constrained by crystal contacts and stabilized by interdomain interaction surfaces, the gross structure of calcium-depleted G4-G6 remains that of the activated form. However, high-resolution details of changes in the ionbinding sites may represent the initial steps toward restoration of the arrangement of domains found in the calcium-free inactive form of gelsolin in solution. Furthermore, bathing crystals with the trivalent calcium ion mimic, Tb 3C , results in anomalous scattering data that permit unequivocal localization of terbium ions in each of the proposed type I and type II ionbinding sites of both halves of gelsolin. In contrast to predictions based on solution studies, we find that no calcium ion is immune to exchange.

Global Structure Changes Associated with Ca2+ Activation of Full-length Human Plasma Gelsolin

“…These dynamic light scattering studies concluded that, in solution, the compact Ca 2ϩ -free gelsolin molecule opens upon binding Ca 2ϩ following a sigmoidal relationship, which is reversible and in which 50% "activation" occurs with 30 nM Ca 2ϩ , whereas a maximum R h is reached at ϳ1 M Ca 2ϩ (18). This observed reversibility is also supported by recent EGTA chelation studies on different crystalline forms of the Ca 2ϩ -activated N-and C-terminal halves of gelsolin whereby the subdomains rearranged toward the compact inactive form (20).…”

Novel interactors and a role for supervillin in early cytokinesis

“…Sequences were oriented and colored using Swiss-PdbViewer (DeepView), v4.0 (http://spdbv.vital-it.ch/). Supervillin amino acids 1326–1699 were automatically aligned with gelsolin repeats 4 through 6 (G4–G6, amino acids 445–765; PDB: 2FH1B) (Chumnarnsilpa et al 2006). Additional structural elements in supervillin residues 1019–1306 were aligned with gelsolin repeats 2 and 3 (G2–G3) and the G3–G4 linker (gelsolin amino acids 189–444; PDB: 2FGHA) (Urosev et al 2006) in forced comparisons.…”

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

“…Surprisingly, then, the crystal structure of equine G1-G3 in a complex with actin (9) shows the type 2 site in G2 to be incompletely formed and devoid of Ca 2ϩ . That this locale within G2 can be induced to form a valid metal ion-binding site is verified by the ability of Tb 3ϩ ions to soak into crystals of the complex of equine G1-G3 with actin and occupy the site (8). Furthermore, a Cd 2ϩ is found in the structure of isolated human G2, coordinated by the side chains of Asp-187, Glu-209, and Asp-259 (10).…”

“…Sequence and structural data (7)(8)(9)(10) suggest that activated actin-bound gelsolin has eight Ca-binding sites. Two, associated with G1 and G4, are identified as type 1, with the metal ion coordinated by residues from both actin and gelsolin (7).…”