Abstract
This is the first report describing temperature based initiation of gelsolin’s F-actin depolymerization activity, even in absence of free Ca2+ or low pH. Small angle X-ray scattering (SAXS) and circular dichroism (CD) studies revealed that temperature in the range of 30–40 °C is capable of opening the G1 domain alone, as remaining domains are held together by the Ca2+-sensitive C-tail latch without any loss in the secondary structural content. Full opening of all domains of tail-less gelsolin, and retention of closed shape for G2–G6 gelsolin merely by heating, further substantiated our findings. The Ca2+/pH independent activity of gelsolin near physiological temperature brought out a query: whether gelsolin is always active, and if not, what might deactivate it? Earlier, PIP2 has been reported to render gelsolin inactive with no structural insight. Reduction in shape parameters and modeling revealed that PIP2 reverses the temperature induced extension of g1-g2 linker leading to a compact shape seen for Ca2+-free gelsolin. Similar results for partially activated gelsolin (by low pH or Ca2+ ions below 0.1 μM) imply that inside cells, depolymerization, capping, and nucleation of F-actin by gelsolin is regulated by the culmination of local Ca2+ ion concentration, pH, temperature and PIP2 levels.
Highlights
Shape-function studies on the six domain actin-assembly regulating protein, gelsolin has brought forth interesting findings, how the compactly packed domains open up completely upon Ca2+ ions binding or partially by sensing low pH1–4
We showed in this work that in absence of these two factors, increase in temperature to 35–40 °C can open and stabilize G1 domain away from the other five locked domains analogous to that induced by low pH
Based on our previous work and complementary work from other groups, there was a query that in physiology, in absence of Ca2+ ions or low pH, gelsolin adopts an inactive resting shape? Our present shape information in conjunction with results reported earlier indicate that very likely, at physiological temperature, intracellular gelsolin exists in this partially open form and the inactive compact shape may never exist except in availability of PIP2 which binds to g1–g2 linker and induces shape changes which bring G1 domain close to G2 domain
Summary
Shape-function studies on the six domain actin-assembly regulating protein, gelsolin has brought forth interesting findings, how the compactly packed domains open up completely upon Ca2+ ions binding or partially by sensing low pH1–4. We found only one study which experimentally compared the effect of temperature on functionality of gelsolin, i.e. how much Ca2+ ions are required by gelsolin to exhibit F-actin depolymerization activity[6] These authors reported that at 37 °C, Ca2+-gelsolin depolymerization of pyrene labelled F-actin was detectable at 0.5 μM Ca2+ which reached half maximal at 2.2 μM Ca2+ and kept increasing till 0.1 mM Ca2+. Their results observed at 24 °C correlated well with previous reports done under similar conditions This ten-fold difference in Ca2+ requirement at 24 vs 37 °C clearly indicated that there is a role of increase in temperature in stabilizing the F-actin binding and depolymerizing competent conformations of gelsolin which overrides/complements the Ca2+-induced effects, though the authors mainly connected their findings to the role of Ca2+-sensitive C-tail region of gelsolin in enabling functional shape of full-length gelsolin and its variants. Experiments were repeated to confirm the observations followed by shape analysis using SAXS experiments which brought forward that the essential and minimal step to be depolymerization competent i.e. opening of G1 domain of gelsolin can be achieved by increase in temperature
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