title: Dissecting the gelsolin-polyphosphoinositide interaction and engineering of a polyphosphoinositide-sensitive gelsolin C-terminal half protein.
abstract: Gelsolin and other proteins in the villin/gelsolin family are regulated by polyphosphoinositides (PPIs), and manipulation of cellular PIP(2) levels alters the structure of the actin cytoskeleton coincident with the dissociation of gelsolin-actin complexes. This work explores the structure-function relationship of the gelsolin-PPI interaction. Circular dichroism experiments show that upon binding to PPIs, the PPI-sensitive N-terminal half of gelsolin undergoes significant secondary and tertiary structural changes that do not occur in the structurally homologous but PPI-insensitive C-terminal half. Secondary structure modeling algorithms predict an alpha-helical conformation for one of the gelsolin PPI-binding sites, P2, which differs from the conformation of P2 in the structure of gelsolin determined by X-ray crystallography, whereas structure prediction of the C-terminal homolog of P2 agrees well with the X-ray crystallography structure. Simulation of a change to helical conformation for P2 using molecular modeling indicates that such a structural transition will destabilize the F-actin-binding sites in domain 2. A hypothesis is proposed that PPIs initiate conformational changes at the PPI-binding site(s) that destabilize the protein structure, and subsequently disrupt the actin-binding sites. To further evaluate the role of P2 in the gelsolin-PPI interaction, a Ct mutant P2Ct is constructed by inserting P2 in place of its C-terminal homologous site. P2Ct interacts with actin in the same way as the wild-type protein. In contrast to Ct, however, P2Ct interacts strongly with PPIs, and its monomeric actin-binding activity becomes regulated by PPIs. It is concluded that the P2 site is sufficient for PPI-sensitivity in gelsolin. Furthermore, the P2 site in P2Ct and the actin-binding sites of Ct do not overlap, suggesting that PPIs regulate actin binding of P2Ct through induction of structural changes, rather than through direct competition.
authors.affiliation: Institute for Medicine and Engineering, University of Pennsylvania, 1010 Vagelos Laboratories, 3340 Smith Walk, Philadelphia 19104, USA. wxian@mail.med.upenn.edu
authors.forename: Wujing
authors.initials: W
authors.lastname: Xian
authors.forename: Paul A
authors.initials: PA
authors.lastname: Janmey
mesh_terms: D000199:Actins / Q000737:chemistry; D000595:Amino Acid Sequence; D001665:Binding Sites; D002413:Cations, Divalent; D018260:Gelsolin / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000737:chemistry*; D018129:Phosphatidylinositol Phosphates / Q000737:chemistry; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012685:Sepharose
chemical_list: D000199:Actins; D002413:Cations, Divalent; D018260:Gelsolin; D010446:Peptide Fragments; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; C098726:gelsolin (150-169); D012685:Sepharose