ELUCIDATION OF THE POLY-L-PROLINE BINDING-SITE IN ACANTHAMOEBA PROFILIN-I BY NMR-SPECTROSCOPY

The multifunctional protein profilin is one of the most abundant prote ins in the cytoplasm and is thought to regulate actin assembly and the phosphoinositide signaling pathway. Profilin binds to several differe nt ligands including actin, poly-L-proline, and the head groups of pol yphosphoinositides. Knowledge of profilin/ligand interactions is impor tant for understanding the physiology of profilin in the cell. As a fi rst step in the characterization of profilin/ligand complexes, we have studied a profilin/poly-L-proline complex in solution using high reso lution NMR spectroscopy. Analysis of profilin NOE's and chemical shift data indicates that the protein secondary structure is conserved upon binding to poly-L-proline and that the binding site is located betwee n the N- and C-terminal helices in a region rich in highly conserved a romatic sidechains. This site is adjacent to the proposed binding site for actin. In addition, the rate constant for dissociation of the com plex is found to be 1.6 +/- 0.2 x 10(4) s(-1).