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Biochemistry. 2000 Oct 10;39(40):12181-9.

The competitive interaction of actin and PIP2 with actophorin is based on overlapping target sites: design of a gain-of-function mutant.

Author information

1
Department of Biochemistry, Ghent University, and Flanders Interuniversity Institute for Biotechnology (VIB), Ledeganckstraat 35, 9000 Gent, Belgium.

Abstract

We studied the effect of mutations in an alpha-helical region of actophorin (residues 91-108) on F-actin and PIP(2) binding. As in cofilin, residues in the NH(2)-terminal half of this region are involved in F-actin binding. We show here also that basic residues in the COOH-terminal half of the region participate in this interaction whereby we extend the previously defined actin binding interface [Lappalainen, P., et al. (1997) EMBO J. 16, 5520-5530]. In addition, we demonstrate that some of the lysines in this alpha-helical region in actophorin are implicated in PIP(2) binding. This indicates that the binding sites of F-actin and PIP(2) on actophorin overlap, explaining the mutually exclusive binding of these ligands. The Ca(2+)-dependent F-actin binding of a number of actophorin mutants (carrying a lysine to glutamic acid substitution at the COOH-terminal positions of the actin binding helical region) may mimic the behavior of members of the gelsolin family. In addition, we show that PIP(2) binding, but not actin binding, of actophorin is strongly enhanced by a point mutation that leads to a reinforcement of the positive electrostatic potential of the studied alpha-helical region.

PMID:
11015196
DOI:
10.1021/bi000816c
[Indexed for MEDLINE]

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