title: Canonical and Noncanonical Sites Determine NPT2A Binding Selectivity to NHERF1 PDZ1.
abstract: Na+/H+ Exchanger Regulatory Factor-1 (NHERF1) is a scaffolding protein containing 2 PDZ domains that coordinates the assembly and trafficking of transmembrane receptors and ion channels. Most target proteins harboring a C-terminus recognition motif bind more-or-less equivalently to the either PDZ domain, which contain identical core-binding motifs. However some substrates such as the type II sodium-dependent phosphate co-transporter (NPT2A), uniquely bind only one PDZ domain. We sought to define the structural determinants responsible for the specificity of interaction between NHERF1 PDZ domains and NPT2A. By performing all-atom/explicit-solvent molecular dynamics (MD) simulations in combination with biological mutagenesis, fluorescent polarization (FP) binding assays, and isothermal titration calorimetry (ITC), we found that in addition to canonical interactions of residues at 0 and -2 positions, Arg at the -1 position of NPT2A plays a critical role in association with Glu43 and His27 of PDZ1 that are absent in PDZ2. Experimentally introduced mutation in PDZ1 (Glu43Asp and His27Asn) decreased binding to NPT2A. Conversely, introduction of Asp183Glu and Asn167His mutations in PDZ2 promoted the formation of favorable interactions yielding micromolar KDs. The results describe novel determinants within both the PDZ domain and outside the canonical PDZ-recognition motif that are responsible for discrimination of NPT2A between two PDZ domains. The results challenge general paradigms for PDZ recognition and suggest new targets for drug development.
authors.affiliation: Department of Pharmacology & Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America.
authors.forename: Tatyana
authors.initials: T
authors.lastname: Mamonova
authors.affiliation: Department of Pharmacology & Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America.
authors.forename: Qiangmin
authors.initials: Q
authors.lastname: Zhang
authors.affiliation: Department of Chemistry and Biochemistry, City College of New York, Chemistry and Biochemistry PhD program, CUNY, New York, New York, United States of America.
authors.forename: Jahan Ali
authors.initials: JA
authors.lastname: Khajeh
authors.affiliation: Department of Chemistry, City College of New York, New York, New York, United States of America.
authors.forename: Zimei
authors.initials: Z
authors.lastname: Bu
authors.affiliation: Department of Pharmacology & Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Bisello
authors.affiliation: Department of Pharmacology & Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America; Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America.
authors.forename: Peter A
authors.initials: PA
authors.lastname: Friedman
mesh_terms: D001665:Binding Sites; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D054731:PDZ Domains / Q000235:genetics; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry* / Q000378:metabolism*; D050613:Sodium-Phosphate Cotransporter Proteins, Type IIa / Q000737:chemistry* / Q000378:metabolism*; D013329:Structure-Activity Relationship
chemical_list: D010455:Peptides; D010750:Phosphoproteins; D017923:Sodium-Hydrogen Exchangers; D050613:Sodium-Phosphate Cotransporter Proteins, Type IIa; C093376:sodium-hydrogen exchanger regulatory factor