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

L. Feng, M. Mejillano, H. L. Yin, J. Chen, G. D. Prestwich

Research output: Contribution to journalArticle

32 Scopus citations

Abstract

Gelsolin, an actin and phosphoinositide binding protein, was photoaffinity labeled using a variety of benzophenone-containing phosphoinositide polyphosphate analogues. The N-terminal half and the C-terminal half of gelsolin showed synergy in the binding of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. Competitive displacement experiments with dibutyryl, dioctanoyl, or dipalmitoyl derivatives of PtdIns(4,5)P2 suggested that, in addition to 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)P2 binding site in the N-terminal half of gelsolin, a new binding site was identified in the C-terminal half by mapping the photocovalently modified peptide fragments. Moreover, increasing concentrations of Ca2+ decreased the binding of the photolabile analogues to the C-terminal phosphoinositide binding site on gelsolin. A molecular model of the binding of PtdIns(4,5)P2 within two folded repeats of gelsolin has been calculated using these data.

Original languageEnglish (US)
Pages (from-to)904-913
Number of pages10
JournalBiochemistry
Volume40
Issue number4
DOIs
StatePublished - Jan 30 2001

ASJC Scopus subject areas

  • Biochemistry

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