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

We studied the effect of mutations in an alpha-helical region of actophorin (residues 91-108) on F-actin and PIP2 binding. As in cofilin, residues in the NH2-terminal half of this region are involved in F-actin binding. We sh ow here also that basic residues in the COOH-terminal half of the region pa rticipate in this interaction whereby we extend the previously defined acti n 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 PIP2 binding. This indicates that t he binding sites of F-actin and PIP2 on actophorin overlap, explaining the mutually exclusive binding of these ligands. The Ca2+-dependent F-actin bin ding of a number of actophorin mutants (carrying a lysine to glutamic acid substitution at the COOH-terminal positions of the actin binding helical re gion) may mimic the behavior of members of the gelsolin family. In addition , we show that PIP2 binding, but not actin binding, of actophorin is strong ly enhanced by a point mutation that leads to a reinforcement of the positi ve electrostatic potential of the studied alpha-helical region.