Full-contact domain labeling: Identification of a novel phosphoinositide binding site on gelsolin that requires the complete protein

Gelsolin, an actin and phosphoinositide binding protein, was photoaffinity labeled using a variety of benzophenone-containing phosphoinositide polypho sphate analogues. The N-terminal half and he C-terminal half of gelsolin sh owed synergy in the binding of phosphatidylinositol 3,5-bisphosphate [PtdIn s(4,5)P-2]. Competitive displacement experiments with dibutyryl, dioctanoyl , or dipalmitoyl derivatives of PtdIns(4,5)Pz suggested that, in addition t o the inositol headgroup, a diacylglyceryl moiety was important for binding ; these analogues also inhibited the gelsolin-severing activity of F-actin. In addition to the previously identified PtdIns(4,5)Pz binding site in the N-terminal half of gelsolin, a new binding site was identified in the C-te rminal half by mapping the photocovalently modified peptide fragments. More over, increasing concentrations of Ca2+ decreased the binding of the photol abile analogues to the C-terminal phosphoinositide binding site on gelsolin . A molecular model of the binding nf PtdIns (4,5)P-2 within two folded rep eats of gelsolin has been calculated using these data.