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Structural studies of the regulation of gelsolin by small ligands

University of British Columbia

Gelsolin regulates the dynamic rearrangement of the actin cytoskeleton by severing actin filaments (F-actin) and capping the newly generated barbed filament ends. Calcium ions and phosphatidylinositol 4,5-bisphosphate (PIP₂), in turn, control these activities. Ca²⁺ -binding within gelsolin primes the protein for binding actin filaments and, during the process, causes drastic rearrangement of its six domains (G1-G6). The significance of specific Ca²⁺ -binding events in this activation process is slowly emerging. A structural basis for the ability of PIP₂ to inhibit ab initio interactions of gelsolin with actin filaments, as well as to induce dissociation of gelsolin already bound to F-actin, remains largely unknown. The reported binding of ATP to gelsolin has been suggested to modulate gelsolin-actin interactions. Association of gelsolin with both of these phosphate-rich molecules is sensitive to the presence of calcium ions. In the projects described in this thesis, I report results of X-ray crystallographic and computational molecular docking experiments to investigate aspects of the regulation of gelsolin by ATP, PIP₂ and calcium ions. Successful introduction of ATP into crystals of inactive gelsolin identify for the first time detailed features of its molecular interactions with gelsolin. Computations confirm the binding of ATP to the observed site to be strong and specific. Demonstration that the ATP-binding site spans both the N- and C-terminal halves of the protein explains the decreased affinity of gelsolin for this ligand in the presence of calcium ions, which induce separation of the halves as part of the activation process. Computational docking experiments suggest residual affinity of activated gelsolin for ATP to be retained at the G2-G3 interface with actin. We propose a model for PIP₂ -binding in the same surface-exposed pocket in gelsolin that is associated with binding ATP phosphates. The model concurs with both the previously reported binding of PIP₂ in this vicinity on gelsolin and the higher affinity of gelsolin for PIP₂ than for ATP. The model, together with the structure of the G1-G3/actin complex, provide insight into the roles of putative PIP₂ -binding sites in both the N- and C-terminal halves of gelsolin. Lastly, exchangeability of metal ions in crystals of G1-G3/actin reflects the transient nature of Ca²⁺ -binding in G2 and helps to explain the loss of local structural stability in a gelsolin mutant that experiences enhanced susceptibility to proteolysis.

Text

2011-02-23

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http://hdl.handle.net/2429/31702

Chemistry

University of British Columbia

Graduate