title: Understanding the role of phosphorylation in the binding mechanism of a PDZ domain.
abstract: The PDZ domain is one of the most common protein-protein interaction domains in mammalian species. While several studies have demonstrated the importance of phosphorylation in interactions involving PDZ domains, there is a paucity of detailed mechanistic data addressing how the PDZ interaction is affected by phosphorylation. Here, we address this question by equilibrium and kinetic binding experiments using PDZ2 from protein tyrosine phosphatase L1 and its interaction with a peptide from the natural ligand RIL. The results show that phosphorylation of a serine residue in the RIL peptide has dual and opposing effects: it increases both the association and dissociation rate constants, which leads to an overall weakening of binding. Furthermore, we performed binding experiments with a RIL peptide in which the serine was replaced by a glutamate, a commonly used method to mimic phosphorylation in proteins. Strikingly, both the affinity and the ionic strength dependence of the affinity differed markedly for the phosphoserine and glutamate peptides. These results show that, in this particular case, glutamate is a poor mimic of serine phosphorylation.
authors.affiliation: Dipartimento di Scienze Biochimiche "A. Rossi Fanelli", Istituto Pasteur Fondazione Cenci Bolognetti, Istituto di Biologia e Patologia Molecolari del CNR, Sapienza Università di Roma, P.le A. Moro 5, 00185 Rome, Italy.
authors.forename: Angelo
authors.initials: A
authors.lastname: Toto
authors.affiliation: Dipartimento di Scienze Biochimiche "A. Rossi Fanelli", Istituto Pasteur Fondazione Cenci Bolognetti, Istituto di Biologia e Patologia Molecolari del CNR, Sapienza Università di Roma, P.le A. Moro 5, 00185 Rome, Italy.
authors.forename: Annalisa
authors.initials: A
authors.lastname: Mattei
authors.affiliation: Department of Medical Biochemistry and Microbiology, Uppsala University, BMC Box 582, SE-75123 Uppsala, Sweden Per.Jemth@imbim.uu.se.
authors.forename: Per
authors.initials: P
authors.lastname: Jemth
authors.affiliation: Dipartimento di Scienze Biochimiche "A. Rossi Fanelli", Istituto Pasteur Fondazione Cenci Bolognetti, Istituto di Biologia e Patologia Molecolari del CNR, Sapienza Università di Roma, P.le A. Moro 5, 00185 Rome, Italy Per.Jemth@imbim.uu.se.
authors.forename: Stefano
authors.initials: S
authors.lastname: Gianni
mesh_terms: D007700:Kinetics; D008958:Models, Molecular; D009994:Osmolar Concentration; D054731:PDZ Domains; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D010455:Peptides; D017027:Protein Tyrosine Phosphatases
keywords: ionic strength; kinetics; peptide binding; protein–protein interaction