Visualizing Temperature Mediated Activation of Gelsolin and Its Deactivation By Pip2: A Saxs Based Study

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.

Antioxidant and Wound Healing Property of Gelsolin in 3T3-L1 Cells

“…Chemicals, Drugs, and Reagents. Methods of expression, purification, and systematic characterization of rhuGSN were followed as described by us earlier [22][23][24]. Briefly, His-tag at N-terminal bearing gelsolin was expressed in E. coli in inducible format.…”

Structural characterization ofMyxococcus xanthusMglC, a component of polarity control system, and its interactions with MglB

“…Cleft 1 is largest among all and is formed at the interface of the dimer with a volume of ~3946 Å 3 . This cleft 1 includes highly conserved positively charged residues imparting a net positive charge to the region as revealed by the ConSurf analysis and electrostatic potential map, respectively (Figure 3B, 3C) (Ashkenazy et al, 2016;Ashkenazy et al, 2010;Baker et al, 2001;Celniker et al, 2013;Glaser et al, 2003;Landau et al, 2005). The cleft 2 and 2′ (prime is used for identical cleft on another protomer in dimer) includes 'FDI' region shown to be involved in binding MglB and has a volume of ~580 Å 3 .…”

“…Considering the presence of flexible or loosely oriented segments, and domains in our protein shapes, we employed chain-ensemble modelling protocol to restore shapes using the SAXS data and deduced parameters using GASBOR program (Svergun et al, 2001). Earlier too, this methodology has been employed to decipher domain-linker shapes (Badmalia et al, 2017;Pandey et al, 2014;Peddada et al, 2013). For GASBOR program, additional inputs were the number of dummy residues (DR) to be used for computing the shape, and were used equal to dimeric state in case of applying P1 symmetry and equal to monomer when considering P2 symmetry.…”

“…Poisson-Boltzmann Solver (APBS) plugin (Baker et al, 2001). For modeling of conserved residues on MglC structure, ConSurf server was used (Ashkenazy et al, 2016;Ashkenazy et al, 2010;Celniker et al, 2013;Glaser et al, 2003;Landau et al, 2005).…”

Myosin-gelsolin cooperativity in actin filament severing and actomyosin motor activity

“…Gelsolin is one of the most abundant and potent actin filament severing, capping and nucleating proteins [8][9][10][11][12][13] among the plethora of ABPs [14,15] that govern remodelling of the actin cytoskeleton in response to various cues [8]. The gelsolin activity is regulated by the complex interplay between calcium (Ca 2+ ), polyphosphoinositide 4, 5-bisphosphate (PIP2) and ATP [16], but it is also partially activated by low pH [17] and high temperature (30-40 °C) [18,19]. Binding of Ca 2+ to several conserved sites on gelsolin, with Ca 2+ affinities ranging from the 100 nM to 10 µM, changes gelsolin structure to facilitate binding to actin and subsequent actin filament severing [11,20] by weakening the noncovalent bonds between actin subunits.…”