Insights into the mechanism of calcium activation of adseverin

Abstract

Actin remodeling is a key step in many cellular processes. Adseverin and gelsolin are calcium dependent actin remodeling proteins in gelsolin superfamily. Gelsolin is involved in cell motility whereas adseverin participates in cell secretion. Gelsolin has a C-terminal extension that forms an a-helix, which covers an F-actin-binding site in the absence of calcium. Equilibrium dialysis experiments suggest that adseverin has one rate-limiting step during activation with respect to actin severing whereas gelsolin has two rate-limiting steps. The second rate-limiting step of gelsolin has been attributed to the unlatching of the C-terminal helix. However, the details of the calcium activation of adseverin and gelsolin remain unclear. Results presented in this study have demonstrated that in solution adseverindisplays different conformations fromgelsolin. However, the calcium induced conformational changes of these proteins are similar. The N-terminal half of adseverin displays an inactive conformation in the absence of calcium that is activated by calcium binding at A3. Calcium binding at the calcium-binding site straightens the long helix that disruptsthe key interactions at the A1:A3 interface (R97-E314 and F64-M310), which is unnecessary for gelsolin. Calcium activation at the C-terminal half of adseverin involves domains rearrangement induced by cooperative calcium binding at A4 and AS but A6. However calcium binding at A6 is key to the activation of the full-length molecule. Moreover actin filament depolymerization assay suggests that the C-terminal half may present actin filament severing activity in the present of calcium. Results in the thesis have provided insights into the details of calcium activation of adseverin providing mechanistic explanations to such questions as, "Why does adseverin require less calcium than gelsolin in inducing severing activity?" "Why is actin filament severing activity by the N-terminus of adseverin calcium dependent whereas it is calcium independent in gelsolin?" Moreover the results have helped explain the common mechanism of calcium activation of the C- terminal half of adseverin and gelsolin.EThOS - Electronic Theses Online ServiceGBUnited Kingdo