[en] 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.
Methods/Findings
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 KD for different ligands. Native mass spectrometry was used as an alternative method for measuring KD values.
Conclusions/Significance
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.
Research Center/Unit :
Laboratoire de Chimie Biologique Structurale, ULiège, Belgium Centre de Biophysique Moléculaire, Orléans, France CIP - Centre d'Ingénierie des Protéines - ULiège
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Tavel, Laurette ; Université de Liège - ULiège > Département de chimie (sciences) > Chimie biologique structurale
Jaquillard, Lucie; Centre National de la Recherche Scientifique - CNRS > CBM-Orleans
Karsisiotis, Andreas; Centre National de la Recherche Scientifique - CNRS > CBM-Orléans
Saab, Fabienne; Centre National de la Recherche Scientifique - CNRS > CBM-Orleans
Jouvensal, Laurence; Centre National de la Recherche Scientifique - CNRS > CBM-Orleans
Brans, Alain ; Université de Liège - ULiège > Centre d'ingénierie des protéines
Delmas, Agnès; Centre National de la Recherche Scientifique - CNRS > CBM-Orleans
Schoentgen, Françoise; Centre National de la Recherche Scientifique - CNRS > CBM-Orleans
Cadene, Martine; Centre National de la Recherche Scientifique - CNRS > CBM-Orleans
Damblon, Christian ; Université de Liège - ULiège > Département de chimie (sciences) > Chimie biologique structurale
Language :
English
Title :
Ligand Binding Study of Human PEBP1/RKIP: Interaction with Nucleotides and Raf-1 Peptides Evidenced by NMR and Mass Spectrometry
Publication date :
27 April 2012
Journal title :
PLoS ONE
eISSN :
1932-6203
Publisher :
Public Library of Science, San Franscisco, United States - California
ANR - Agence Nationale de la Recherche LNCC - Ligue Nationale Contre le Cancer Cancéropôle du Grand Ouest CNRS - Centre National de la Recherche Scientifique ULiège. Patrimoine - Université de Liège. Patrimoine EU - European Union
Funding number :
ANR-08-BLAN-0033; EU-NMR program (RII3-026145)
Funding text :
Régions Bretagne, Centre, Pays de la Loire et Poitou-Charentes (AO2007-2010)
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