PhD Graduate: Shalini Nag
Thesis Title: Structural analysis of calcium-induced changes in gelsolin and adseverin
Microfilaments are dynamic linear assemblies of actin, the synchronized polymerization and depolymerization of which contribute to the force required for eukaryotic cellular movement. More than 300 actin-binding proteins regulate actin function by either altering the kinetics of actin polymerization or mediating the interactions between the actin network and other cellular components. The gelsolin superfamily proteins are calcium-dependent actin modulators that can bind monomeric actin or sever, cap, nucleate and bundle filaments, and thus participate in numerous actin-dependent cellular processes ranging from cell motility and exocytosis. Gelsolin also has key actin-independent functions as an anti-apoptotic protein in the cytosol, and anti-amyloidogenic protein in the plasma and cerebrospinal fluid. Further, a single mutation in gelsolin results in familial amyloidosis of Finnish-type (FAF). Understanding how gelsolin functions in the vastly different environments of the cytoplasm and plasma, and how a single mutation renders this protein amyloidogenic, requires the elucidation of the mechanisms through which calcium binding is translated into large domain movements during activation and function. Determination and analysis of the structures of actin-bound N-terminal half (G1-G3) and calcium-bound domain-3 (G3) of human cytoplasmic gelsolin, in combination with the functional characterization of calcium-binding site mutants of gelsolin, identify the contribution of various domains to activation. Further, the structure of a gelsolin homologue from zebrafish, scinderin-like B, offers the first view of a partially activated full-length gelsolin family protein and provides key insights into the mechanisms of calcium- and pH-induced activation. Finally, functional comparison of gelsolin with adseverin, its closest vertebrate homologue, reveals common functional mechanisms and highlights regulatory differences between them. While these findings are generally relevant to the cellular functions of gelsolin and adseverin, they have specific implications for gelsolin’s role in apoptosis and FAF.
The key role of domain 2 in gelsolin activation has implications for Familial-amyloidosis (Finnish-type).
A) Structure of inactive full-length human gelsolin (PDB ID 3FFN). B) Structure of human gelsolin N-terminal half in complex with actin. Black spheres represent the bound calcium ions. (This study, PDB ID 3FFK). The black arrow marks the Furin-cleavage site within G2, that is protected in both the inactive (A) and active (B) conformations. C) Actin depolymerization assay of gelsolin calcium-binding site mutants in EGTA. Full-length gelsolin (GFL), and mutants with substitutions only within G2 or G6 have no activity (only one mutant, D2E2, is displayed for clarity). The gelsolin mutant lacking the C-terminal tail helix (GTL) has calcium-independent actin depolymerization activity. Cooperation between domains 2 and 6 (three-site mutants - D2E2D6, D2D6E6 and four-site mutant – D2E2D6E6) results in calcium-independent actin-depolymerization activity. Thus, mutation in domain 2 reduces the requirement for calcium during activation and destabilizes the inactive structure, making gelsolin susceptible to cleavage by Furin.
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