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

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 [Ca2+] >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 [Ca2+], the apparent size of the C-terminal half as determined by gel filtration was reduced, indicating a transition into a more compact conformation. Moreover, Ca2+ inhibited the cleavage by trypsin at Lys 634 within the loop connecting segments 5 and 6. Though the inhibitory effect was observed already at [Ca2+] of 10-7 M, it was enhanced with increasing [Ca2+], attaining saturation only at >10-4 M Ca2+. This indicates that the initial conformational changes are followed by additional molecular transitions in the range of 10-5−10-4 M [Ca2+]. 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.