Co‐operation of domain‐binding and calcium‐binding sites in the activation of gelsolin

Abstract

Gelsolin is an abundant calcium dependent actin filament severing and capping protein. In the absence of calcium the molecule is compact but in the presence of calcium, as its six similar domains alter their relative position, a generally more open configuration is adopted to reveal the three actin binding sites. It is generally held that a Ôhelical-latchÕ at the C-terminus of gelsolin's domain 6 (G6), binds domain 2 (G2) to keep gelsolin in the calcium-free compact state, and that the crutial calcium binding site(s) reside in the C-terminal half of gelsolin perhaps involving the C-terminal helix itself has to be bound to release this latch. Here we provide evidence for a calcium dependent conformational change within G2 (K d ¼ 15 lM). We also report a calcium dependent binding site for the C-terminus (G4-6) within G2 and delimit this further to a specific region formed by residues 203-225 and 159-193. It is known that the activation of gelsolin involves multiple calcium binding events (around 6) the first of which (in G6) may release the latch. We propose that the calcium-dependent conformational change in G2 may be a subsequent step that is necessary for the dissociation of G2 from G4-6, and that this movement occurs in sympathy with calcium induced conformational changes within G6 by the physical coupling of the two calcium binding sites within G2 and G6. Additional calcium binding in other domains then result in the complete opening and activation of the gelsolin molecule.

Structural characterization of Salmonella typhimurium YeaZ, an M22 O‐sialoglycoprotein endopeptidase homolog

“…As an extension of this, some HALF proteins have also been identified where the metal ion and nucleotide binding sites are only partly formed by the HALF partner. Formation of the complete binding site thus requires additional residues presented on the surface of a different protein; examples include the actin‐gelsolin complex and glycerol kinase 53, 54. Analysis of the YeaZ crystal lattice packing suggests a “crescent”‐shaped dimer, an unusual arrangement for a HALF protein.…”

Gelsolin: The tail of a molecular gymnast

“…Inactive gelsolin undergoes either two or three conformational changes as calcium concentrations rise from 10 nM to ∼5 μM, as a result of binding of two or three calcium ions with K d values estimated to be 0.1 and/or 0.3 μM and 6.4 μM [Kinosian et al, ; Lin et al, ]. These events may reflect the release of the tail latch [Kwiatkowski et al, ; Pope et al, ; Kinosian et al, ; Lin et al, ; Lueck et al, ; Choe et al, ; Kolappan et al, ; Lagarrigue et al, ,b], for which recent evidence [Nag et al, ] implicates a cooperative pair of calcium‐binding events at the type‐2 sites in G2 and G6. Indeed, sequence analysis reveals that human gelsolin superfamily proteins that lack a full complement of six type‐2 sites still retain a type‐2 site in domain 2 (supervillin) or domain 6 (flightless I), testifying to the importance of breaking the domain 2:domain 6 interactions during activation (Fig.…”

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

“…In addition to the above mentioned regulation of gelsolin activity by Ca 2ϩ ions and PIP2, acidic pH (ϳ6) has also been shown to enable actin assembly/disassembly function of gelsolin (12)(13)(14). It was shown that even when intracellular Ca 2ϩ concentrations were about subnanomolar range, pH value close to 6 allowed gelsolin/G-actin binding as estimated by an increase in fluorescence intensity of labeled actin, with the rate of formation of the complex being about five times faster at pH 6 than that at pH 8 (12).…”