title: Probing the phosphoinositide 4,5-bisphosphate binding site of human profilin I.
abstract: 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)P2). The P-1-tethered D-myoinositol 1,4,5-trisphosphate (Ins(1,4,5)P3) modified profilin I efficiently and specifically; the covalent labeling could be displaced by co-incubation with an excess of PtdIns(4,5)P2 but not with Ins(1,4,5)P3. The acyl-modified PtdIns(4,5)P2 analog showed little protein labeling even at very low concentrations, whereas the head-group-modified PtdIns(4,5)P2 phosphotriester-labeled monomeric and oligomeric profilin. Mass spectroscopic analyses of CNBr digests of [3H]BZDC-Ins(1,4,5)P3-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)P2-binding site of profilin I includes a bisphosphate interaction with a base-rich motif in the carboxy-terminal helix and contact between the lipid moiety of PtdIns(4,5)P2 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.
authors.affiliation: Department of Chemistry, University at Stony Brook, New York 11794-3400, USA.
authors.forename: A
authors.initials: A
authors.lastname: Chaudhary
authors.forename: J
authors.initials: J
authors.lastname: Chen
authors.forename: Q M
authors.initials: QM
authors.lastname: Gu
authors.forename: W
authors.initials: W
authors.lastname: Witke
authors.forename: D J
authors.initials: DJ
authors.lastname: Kwiatkowski
authors.forename: G D
authors.initials: GD
authors.lastname: Prestwich
mesh_terms: D001665:Binding Sites; D003285:Contractile Proteins; D006801:Humans; D008840:Microfilament Proteins / Q000737:chemistry / Q000378:metabolism*; D015335:Molecular Probes; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000737:chemistry / Q000378:metabolism*; D051304:Profilins; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D003285:Contractile Proteins; D008840:Microfilament Proteins; D015335:Molecular Probes; C495738:PFN1 protein, human; D019269:Phosphatidylinositol 4,5-Diphosphate; D051304:Profilins; D011994:Recombinant Proteins