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

Abstract

Gelsolin is a key actin cytoskeleton-modulating protein primarily regulated by calcium and phosphoinositides. In addition, low pH has also been suggested to activate gelsolin in the absence of Ca 2؉ ions, although no structural insight on this pathway is available except for a reported decrement in its diffusion coefficient at low pH. We also observed ϳ1.6-fold decrease in the molecular mobility of recombinant gelsolin when buffer pH was lowered from 9 to 5. Analysis of the small angle x-ray scattering data collected over the same pH range indicated that the radius of gyration and maximum linear dimension of gelsolin molecules increased from 30.3 to 34.1 Å and from 100 to 125 Å , respectively. Models generated for each dataset indicated that similar to the Ca 2؉ -induced process, low pH also promotes unwinding of this six-domain protein but only partially. It appeared that pH is able to induce extension of the G1 domain from the rest of the five domains, whereas the Ca 2؉ -sensitive latch between G2 and G6 domains remains closed. Interestingly, increasing the free Ca 2؉ level to merely ϳ40 nM, the partially open pH 5 shape "sprung open" to a shape seen earlier for this protein at pH 8 and 1 mM free Ca 2؉ . Also, pH alone could induce a shape where the g3-g4 linker of gelsolin was open when we truncated the C-tail latch from this protein.Our results provide insight into how under physiological conditions, a drop in pH can fully activate the F-actin-severing shape of gelsolin with micromolar levels of Ca 2؉ available.

Global Shapes of F-actin Depolymerization-competent Minimal Gelsolins

“…The structure of the F-actin depolymerization-competent 25-160-residue fragment of gelsolin in complex with the actin supported the hypothesis that the partial G2 domain binds along the side of the actin filament in such a way that it targets the G1 domain to intercalate between the actin subunit bound to G2 and the adjacent actin subunit and to rupture the noncovalent interactions holding them together (18). The opening of the G1 domain from rest of the molecule might thus be a prerequisite for the F-actin depolymerization function of gelsolin as has also been supported by the recent studies (3,4).…”

“…2A), which was repeatedly observed in the absence of any gelsolin variant and could be due to the dilution of F-actin from 4 M to 100 nM concentration just prior to the fluorescence measurements (as described under "Materials and Methods"). Because the G1 domain and the native g1-g2 linker are essential for the F-actin depolymerizing ability of GSN (3,4,17), as expected, the addition of G2-G6 and G4-G6 (data not shown) to labeled F-actin in the buffer containing 1 mM of free Ca 2ϩ at pH 8 did not lower the relative fluorescence ( Fig. 2A).…”

“…Cloning of the full-length gelsolin (GSN) has been described earlier (4). The plasmids harboring the genes for recombinant gelsolin mutants G1-G3 (residues 1-371), G1-G2 (residues 1-241), G2-G6 (residues 158 -755), G2-G3 (residues 158 -371), G4-G6 (residues 412-755), dT-GSN (residues 1-731), 1-161, 25-161, 28 -161, 30 -161, 32-161, 34 -161, 36 -161, 40 -161, 42-161, 56 -161, 25-158 and 25-156) were created by PCR amplification of the corresponding fragments using specific primers and pET303/gelsolin as template.…”

“…These isoforms exhibit a compact globular structure under Ca 2ϩ -free conditions at the physiological pH and attain an open functionally active structure in the presence of 1 mM Ca 2ϩ or at lower pH (2)(3)(4). Radiolytic footprinting and small angle x-ray scattering (SAXS) 4 studies have elucidated a three-stage opening of the gelsolin molecule upon sequential binding of free Ca 2ϩ .…”

“…These isoforms exhibit a compact globular structure under Ca 2ϩ -free conditions at the physiological pH and attain an open functionally active structure in the presence of 1 mM Ca 2ϩ or at lower pH (2)(3)(4). Radiolytic footprinting and small angle x-ray scattering (SAXS) 4 studies have elucidated a three-stage opening of the gelsolin molecule upon sequential binding of free Ca 2ϩ . The first transition from the inactive molecule to the capping and severing-competent intermediate state is achieved primarily by the opening of the tail latch at a Ca 2ϩ concentration of ϳ5 M, although further opening occurs at 100 M Ca 2ϩ by the unwinding of the g3-g4 linker (3,5).…”