Probing the phosphoinositide 4,5-bisphosphate binding site of human profilin I

Background: Profilin is a widely and highly expressed 14 kDa protein that binds actin monomers, poly(L-proline) and polyphosphoinositol lipids. It participates in regulating actin-filament dynamics that are essential for many types of cell motility, We sought to investigate the site of interaction of profilin with phosphoinositides. Results: Human profilin I was covalently modified using three tritium-labeled 4-benzoyldihydrocinnamoyl (BZDC)-containing photoaffinity analogs of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P-2). The P-1-tethered D-myoinositol 1,4,5-trisphosphate (Ins(1,4,5)P-3) modified profilin I efficiently and specifically; the covalent labeling could be displaced by co-incubation with an excess of PtdIns(4,5)P-2 but not with Ins(1,4,5)P-3. The acyl-modified PtdIns(4,5)P-2 analog showed little protein labeling even at very low concentrations, whereas the head-group-modified PtdIns(4,5)P-2 phosphotriester-labeled monomeric and oligomeric profilin. Mass spectroscopic analyses of CNBr digests of [H-3]BZDC-Ins(1 ,4,5)P-3-modified recombinant profilin suggested that modification was in the amino-terminal helical CNBr fragment. Edman degradation confirmed Ala1 of profilin I (residue 4 of the recombinant protein) was modified. Molecular models show a minimum energy conformation in which the hydrophobic region of the ligand contacts the amino-terminal helix whereas the 4,5-bisphosphate interacts with Arg135 and Arg136 of the carboxy-terminal helix. Conclusions: The PtdIns(4,5)P-2-binding site of profilin 1 includes a bisphosphate interaction with a base-rich motif in the carboxy-terminal helix and contact between the lipid moiety of PtdIns(4,5)P-2 and a hydrophobic region of the aminoterminal helix of profilin. This is the first direct evidence for a site of interaction of the lipid moiety of a phosphoinositide bisphosphate analog with profilin.