title: Ligand binding study of human PEBP1/RKIP: interaction with nucleotides and Raf-1 peptides evidenced by NMR and mass spectrometry.
abstract: BACKGROUND
  Human Phosphatidylethanolamine binding protein 1 (hPEBP1) also known as Raf kinase inhibitory protein (RKIP), affects various cellular processes, and is implicated in metastasis formation and Alzheimer's disease. Human PEBP1 has also been shown to inhibit the Raf/MEK/ERK pathway. Numerous reports concern various mammalian PEBP1 binding ligands. However, since PEBP1 proteins from many different species were investigated, drawing general conclusions regarding human PEBP1 binding properties is rather difficult. Moreover, the binding site of Raf-1 on hPEBP1 is still unknown.
  
  
  RESULTS
  In the present study, we investigated human PEBP1 by NMR to determine the binding site of four different ligands: GTP, FMN, and one Raf-1 peptide in tri-phosphorylated and non-phosphorylated forms. The study was carried out by NMR in near physiological conditions, allowing for the identification of the binding site and the determination of the affinity constants K(D) for different ligands. Native mass spectrometry was used as an alternative method for measuring K(D) values.
  
  
  CONCLUSIONS
  Our study demonstrates and/or confirms the binding of hPEBP1 to the four studied ligands. All of them bind to the same region centered on the conserved ligand-binding pocket of hPEBP1. Although the affinities for GTP and FMN decrease as pH, salt concentration and temperature increase from pH 6.5/NaCl 0 mM/20°C to pH 7.5/NaCl 100 mM/30°C, both ligands clearly do bind under conditions similar to what is found in cells regarding pH, salt concentration and temperature. In addition, our work confirms that residues in the vicinity of the pocket rather than those within the pocket seem to be required for interaction with Raf-1.
authors.affiliation: Department of Chemistry, University of Liège, Liège, Belgium.
authors.forename: Laurette
authors.initials: L
authors.lastname: Tavel
authors.forename: Lucie
authors.initials: L
authors.lastname: Jaquillard
authors.forename: Andreas I
authors.initials: AI
authors.lastname: Karsisiotis
authors.forename: Fabienne
authors.initials: F
authors.lastname: Saab
authors.forename: Laurence
authors.initials: L
authors.lastname: Jouvensal
authors.forename: Alain
authors.initials: A
authors.lastname: Brans
authors.forename: Agnès F
authors.initials: AF
authors.lastname: Delmas
authors.forename: Françoise
authors.initials: F
authors.lastname: Schoentgen
authors.forename: Martine
authors.initials: M
authors.lastname: Cadene
authors.forename: Christian
authors.initials: C
authors.lastname: Damblon
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D005486:Flavin Mononucleotide / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D013058:Mass Spectrometry; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D009711:Nucleotides / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D049488:Phosphatidylethanolamine Binding Protein / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation; D019908:Proto-Oncogene Proteins c-raf / Q000737:chemistry*; D051381:Rats; D012492:Salts / Q000494:pharmacology; D013696:Temperature
chemical_list: D008024:Ligands; D009711:Nucleotides; C511482:PEBP1 protein, human; D010446:Peptide Fragments; D049488:Phosphatidylethanolamine Binding Protein; D012492:Salts; D005486:Flavin Mononucleotide; D006160:Guanosine Triphosphate; D019908:Proto-Oncogene Proteins c-raf