The activation of gelsolin by low pH

Abstract

Gelsolin is a multidomain and multifunction protein that nucleates the assembly of filaments and severs them. The activation of gelsolin by calcium is a multistep process involving many calcium binding sites that act to unfold the molecule from a tight structure to a more loose form in which three actin‐binding sites become exposed. Low pH is also known to activate gelsolin, in the absence of calcium and this too results in an unfolding of the molecule. Less is known how pH‐activation occurs but we show that there are significant differences in the mechanisms that lead to activation. Crucially, while it is known that the bonds between G2 and G6 are broken by co‐operative occupancy of calcium binding sites in both domains [Lagarrique, E., Maciver, S. K., Fattoum, A., Benyamin, Y. & Roustan, C. (2003) Eur. J. Biochem. 270, 2236–2243.], pH values that activate gelsolin do not result in a weakening of the G2‐G6 bonds. We report the existence of pH‐dependent conformational changes within G2 and in G4–6 that differ from those induced by calcium, and that low pH overrides the requirement for calcium for actin‐binding within G4–6 to a modest extent so that a Kd of 1 µm is measured, compared to 30–40 nm in the presence of calcium. Whereas the pH‐dependent conformational change in G2 is possibly different from the change induced by calcium, the changes measured in G4–6 appear to be similar in both calcium and low pH.

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

“…The compact packing of the G2-G6 domains in the structure solved for pH 5 could be due to the close C-tail latch, which "locks" G2/G6 domains and is sensitive only to Ca 2ϩ ions. This observation corroborates with an earlier report that the opening of the C-tail latch, which is one of the first events of Ca 2ϩ -dependent gelsolin activation, might be absent in the pH-mediated activation process (15).…”

“…Decrement in diffusion coefficient values measured by dynamic light scattering (DLS) supported that lower pH induces opening of the solution structure of gelsolin (13), similar to that seen as a function of increase in Ca 2ϩ ions without changing buffer pH (16). Additionally, biochemical studies supported that low pH leads to a cascade of ion-pair exchanges in gelsolin, which causes disruption of interdomain bonds, but these events were concluded to be distinct from those induced by Ca 2ϩ binding (15). Importantly, one of the first events of Ca 2ϩ -dependent activation, the opening of the C-tail latch (interaction between the C-tail of the G6 and G2 domain), was suggested to be absent in the pH-mediated activation process.…”

“…In addition, they also reported that starting from monomeric G-actin, actin polymerization was accelerated at low pH (ϳ5.3) in the absence of Ca 2ϩ , at levels even higher than those observed at pH 7.4 in the presence of 200 M Ca 2ϩ (13). In contrast, another group proposed that pH induces partial activation because they observed that at pH 5.7, the affinity of the G4-G6 half toward actin was only 1 M in comparison with about 30 nM in buffer containing 1 mM Ca 2ϩ and pH 7.4 (15). Bringing clarity to this debate, our present work provides structural insight into how low pH can open up the g1-g2 linker, which is essential for the F-actin-severing function of gelsolin, although mechanistically speaking, it still remains to be explored how the partially activated structure might sit across/on F-actin and perform its severing action.…”

“…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). Other reports also showed that the lowering of pH (Ͻ6) in the absence of calcium caused activation of gelsolin because with decrement in buffer pH, both filament severing as well as nucleating activities of gelsolin were substantially potentiated (13,15). At a pH Ͻ6.0, the gelsolin activation has been found to require either no Ca 2ϩ or substantially reduced levels of Ca 2ϩ to exhibit weak/moderate activity similar to that observed at Ca 2ϩ concentration of 200 M at pH 7 (13).…”

Structural Basis for pH-mediated Regulation of F-actin Severing by Gelsolin Domain 1

“…Interestingly, the F-actin depolymerization activity of G1+ is dependent on Ca 2+ and pH, similar to the full-length protein but different from the N-terminal half including domains G1-G3. Previous reports have demonstrated that gelsolin requires no Ca 2+ for in vitro binding to G-actin, nucleating F-actin formation, and severing F-actin filaments at a pH less than 6.01213. According to recent small angle X-ray scattering (SAXS) data, lowering pH from 8 to 5 induces the increase of the gyration radii of gelsolin and G1+ in the absence of Ca 2+ by 9.7% and 11.6%, respectively1014.…”