Isolation and characterization of two mutants of human profilin I that do not bind poly(L-proline)

A simple procedure for the isolation of profilin mutants hating a reduced 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-proline), actin, and phosphatidylinositol (4,5)-bisphosphate (PIP2) was studied, Both mutations abolished the poly(L-proline)-binding activity of profilin, This suggests that the arrangement of the N- and C-terminal helices forming the poly(L-proline)-binding site depends on the stabilizing interaction between W3 and W31 in the underlying beta-strand, and that the H133S mutation in the C-terminal helix also must have distorted the arrangement of the terminal helices, Both mutations caused a reduced affinity for actin, with the W3N replacement hating the most pronounced effect, This shows that structural changes in the poly(L-proline)-binding region of profilin can affect the distantly located actin-binding site, Thus, ligands influencing the structure of the poly(L-proline)-binding site may regulate actin polymerization through profilin. This is consonant with the finding that PIP2, which changes the tryptophan fluorescence in wild-type profilin and dissociates the profilin:actin complex in vitro, binds more strongly to the W3N mutant profilin, Thus, the poly(L-proline)-binding surface represents a crucial regulatory site of profilin function. (C) 1997 Federation of European Biochemical Societies.