title: Molecular basis of the PED/PEA15 interaction with the C-terminal fragment of phospholipase D1 revealed by NMR spectroscopy.
abstract: PED/PEA15 is a small protein involved in many protein-protein interactions that modulates the function of a number of key cellular effectors involved in major cell functions, including apoptosis, proliferation and glucose metabolism. In particular, PED/PEA15 interacts with the phospholipase D (PLD) isoforms 1 and 2 increasing protein kinase C-α isoform activity and affects both insulin-stimulated glucose transport and glucose-stimulated insulin secretion. The C-terminal portion (residues 712-1074) of PLD1, named D4, is still able to interact with PED/PEA15. In this study we characterized, by means of NMR spectroscopy, the molecular interaction of PED/PEA15 with D4α, a smaller region of D4, encompassing residues 712-818, shown to have the same affinity for PED/PEA15 and to induce the same effects as D4 in PED/PEA15-overexpressing cells. Chemical shift perturbation (CSP) studies allowed to define D4α binding site of PED/PEA15 and to identify a smaller region likely affected by an allosteric effect. Moreover, ELISA-like experiments showed that three 20-mer overlapping synthetic peptides, covering the 762-801 region of D4α, strongly inhibit PED/PEA15-D4α interaction through their binding to PED/PEA15 with KDs in low micromolar range. Finally, molecular details of the interaction of PED/PEA15 with one of the three peptides have been revealed by CSP and saturation transfer difference (STD) analyses.
authors.affiliation: Istituto di Biostrutture e Bioimmagini, CNR, Napoli, Italy.
authors.forename: Biancamaria
authors.initials: B
authors.lastname: Farina
authors.forename: Nunzianna
authors.initials: N
authors.lastname: Doti
authors.forename: Luciano
authors.initials: L
authors.lastname: Pirone
authors.forename: Gaetano
authors.initials: G
authors.lastname: Malgieri
authors.forename: Emilia M
authors.initials: EM
authors.lastname: Pedone
authors.forename: Menotti
authors.initials: M
authors.lastname: Ruvo
authors.forename: Roberto
authors.initials: R
authors.lastname: Fattorusso
mesh_terms: D051017:Apoptosis Regulatory Proteins; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D010739:Phospholipase D / Q000737:chemistry* / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation
chemical_list: D051017:Apoptosis Regulatory Proteins; D047908:Intracellular Signaling Peptides and Proteins; C092039:PEA15 protein, human; D010446:Peptide Fragments; D010750:Phosphoproteins; D010739:Phospholipase D; C106801:phospholipase D1