Calcium-Induced Conformational Changes in the C-Terminal Half of Gelsolin Stabilize Its Interaction with the Actin Monomer

Abstract

The basic mechanism for the nucleating effect of gelsolin on actin polymerization is the formation of a complex of gelsolin with two actin monomers. Probably due to changes in the C-terminal part of gelsolin, a stable ternary complex is only formed at [Ca(2+)] >10(-5) M [Khaitlina, S., and Hinssen, H. (2002) FEBS Lett. 521, 14-18]. Therefore, we have studied the binding of actin monomer to the isolated C-terminal half of gelsolin (segments 4-6) over a wide range of calcium ion concentrations to correlate the conformational changes to the complex formation. With increasing [Ca(2+)], the apparent size of the C-terminal half as determined by gel filtration was reduced, indicating a transition into a more compact conformation. Moreover, Ca(2+) inhibited the cleavage by trypsin at Lys 634 within the loop connecting segments 5 and 6. Though the inhibitory effect was observed already at [Ca(2+)] of 10(-7) M, it was enhanced with increasing [Ca(2+)], attaining saturation only at >10(-4) M Ca(2+). This indicates that the initial conformational changes are followed by additional molecular transitions in the range of 10(-5)-10(-4) M [Ca(2+)]. Consistently, preformed complexes of actin with the C-terminal part of gelsolin became unstable upon lowering the calcium ion concentrations. These data provide experimental support for the role of the type 2 Ca-binding sites in gelsolin segment 5 proposed by structural studies [Choe et al. (2002) J. Mol. Biol. 324, 691]. We assume that the observed structural transitions contribute to the stable binding of the second actin monomer in the ternary gelsolin-actin complex.

Effects of a surface topography composite with puerariae radix on human STRO-1-positive stem cells

“…These results indicated dynamic polymerizing condition of actin which happened in differentiation state (65) and because gelsolin works in a calcium-dependent manner (39) thus this event supported the up-regulation of annexin V. Furthermore, up-regulation of annexin V was related with the up-regulation of osteocalcin in the cultured cells (56) which was supported by osteocalcin staining. However, changes in expressions of these proteins were not significant.…”

“…Gelsolin is an actin-binding protein that nucleates actin polymerization and severs and caps actin filaments in a calcium dependent manner (39).…”

Calcium Ion Exchange in Crystalline Gelsolin

“…Furthermore, the initial soak conditions, in which calcium was observed in this site, contained calcium levels far below that reported for occupation of this site. 13 Thus, stabilization of the G5-G6 interface appears to increase the affinity of the G5 type II site for calcium. The interaction of G4-G6 with actin may also reasonably be expected to stabilize the G5-G6 interface leading to a modulation of the affinity of this site in the presence of actin.…”

“…A third, low-affinity calcium-binding site in G4-G6, characterized by K d Z100 mM, has also been identified from changes in susceptibility to chemical or enzymatic modification. 13,14 Structural studies have demonstrated that there is a conserved calcium-binding site in each of the six gelsolin domains, referred to as type II binding sites. 15 Hence, biochemical and structural data concur that there are three calcium-binding sites in G4-G6.…”

“…More recently, protease susceptibility studies have demonstrated protection of the loop between G5 and G6 at above 50 mM Ca 2C , leading the authors to conclude that the low-affinity calcium-binding site lies in G5. 13 Here, we probe the calcium-binding sites of the N and C-terminal halves of gelsolin by varying the free metal ion environment surrounding protein crystals. We verify two viable type I and six viable type II metal ion binding sites in the crystalline fragments.…”