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Towards a crystallographic structure for the N-terminal half of gelsolin bound to actin Urosev, Dunja

Abstract

Gelsolin belongs to a family of proteins that participates in the reorganization of cytoskeletal actin, a shared requirement of such processes as cell movement, cell division and apoptosis. Its functions include actin filament nucleation, severing and capping. Gelsolin consists of six structurally analogous domains (G1-G6) that, in the Ca2+-free, inactive state, are packed in such way that none of the actin-binding sites are accessible. Binding of Ca2 + ions to gelsolin causes large changes in the relative positions and orientations of the six domains, resulting in exposure of several actin-binding surfaces, and subsequent binding and severing of actin filaments. Filament end-binding sites have been identified previously in domains Gl and G4, while a distinct filament side-binding site has been attributed to G2. This thesis describes protein preparation and crystallization experiments that led to the structure at 3 A resolution of the N-terminal half of gelsolin (G1-G3) bound to one actin molecule in the presence of Ca2 + ions. The structure reveals for the first time the details of how G2 and G3 interact with the same actin to which Gl is attached. As expected, the changes in the relative orientation of domains within the N-terminal half of gelsolin on activation are large. Previously unidentified contacts between G3 and actin are observed. The existence of a Ca2 + in the type-2 binding site in activated G3, which was inferred by sequence comparison within gelsolin domains, is confirmed. In addition, docking the structure into existing molecular models for an actin filament permits proposal of a self-consistent mechanism for how intact gelsolin is activated, binds to the side of an actin filament, severs and then caps one of the newly cut filament ends.

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