ISOLATION AND CHARACTERIZATION OF 2 MUTANTS OF HUMAN PROFILIN-I THAT DO NOT BIND POLY(L-PROLINE)

A simple procedure for the isolation of profilin mutants hating a redu ced capacity to bind poly(L-proline) is used to isolate two mutants of human profilin I, W3N and H133S, Binding of the mutants to poly(L-pro line), actin, and phosphatidylinositol (4,5)-bisphosphate (PIP2) was s tudied, Both mutations abolished the poly(L-proline)-binding activity of profilin, This suggests that the arrangement of the N- and C-termin al helices forming the poly(L-proline)-binding site depends on the sta bilizing interaction between W3 and W31 in the underlying beta-strand, and that the H133S mutation in the C-terminal helix also must have di storted the arrangement of the terminal helices, Both mutations caused a reduced affinity for actin, with the W3N replacement hating the mos t pronounced effect, This shows that structural changes in the poly(L- proline)-binding region of profilin can affect the distantly located a ctin-binding site, Thus, ligands influencing the structure of the poly (L-proline)-binding site may regulate actin polymerization through pro filin. This is consonant with the finding that PIP2, which changes the tryptophan fluorescence in wild-type profilin and dissociates the pro filin:actin complex in vitro, binds more strongly to the W3N mutant pr ofilin, Thus, the poly(L-proline)-binding surface represents a crucial regulatory site of profilin function. (C) 1997 Federation of European Biochemical Societies.