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 [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.
Institute of Cytology, Russian Academy of Sciences, 196064 St. Petersburg, Russia.
Sofia
S
Khaitlina
Markus
M
Walloscheck
Horst
H
Hinssen
D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002850:Chromatography, Gel; D018260:Gelsolin / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary / Q000187:drug effects; D011817:Rabbits; D013552:Swine; D013696:Temperature; D014357:Trypsin / Q000378:metabolism
D000199:Actins; D018260:Gelsolin; D010446:Peptide Fragments; D014357:Trypsin; D002118:Calcium