title: Structural characterization of the intermolecular interactions of synapse-associated protein-97 with the NR2B subunit of N-methyl-D-aspartate receptors.
abstract: The synapse-associated protein-97 (SAP97) is important in the proper trafficking and cell surface maintenance of the N-methyl-D-aspartate ionotropic glutamate receptor. The molecular scaffold/receptor interaction is mediated by the association of the C terminus of the NR2B subunit of the N-methyl-D-aspartate receptor with the PDZ domains of SAP97. Here, we characterize the binding of the C terminus of NR2B with the PDZ domains of SAP97 and determine the structure of the PDZ1-NR2B complex employing high-resolution NMR. Based on fluorescence anisotropy, the NR2B subunit binds to the first and second PDZ domains of SAP97, with higher affinity for PDZ2; no appreciable binding to PDZ3 could be measured. The structural features of the NR2B bound to PDZ1 is consistent with the canonical PDZ-binding motif with the glutamic acid at the -3 position of the C terminus (i.e. -E-S-D-V) interacting with the beta2/beta3 loop. Two sites within the loop of PDZ1 were replaced with the corresponding residue from PDZ2, D243G and P245Q. The former mutation, designed to remove a possible Coulombic repulsion between E(-3)(NR2B) and Asp-243 (PDZ1) has only a minimal effect on binding. The P245Q mutation leads to a 2-fold increase in binding affinity of NR2B, approaching that observed for wild-type PDZ2. These results indicate that modification of the beta2/beta3 loop provides an avenue for regulating the ligand specificity of PDZ domains.
authors.affiliation: Department of Physics, Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912, USA.
authors.forename: Lei
authors.initials: L
authors.lastname: Wang
authors.forename: Andrea
authors.initials: A
authors.lastname: Piserchio
authors.forename: Dale F
authors.initials: DF
authors.lastname: Mierke
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005454:Fluorescence Polarization; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments; D011485:Protein Binding; D021122:Protein Subunits; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000737:chemistry / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; C501119:Dlg1 protein, rat; D008565:Membrane Proteins; C121001:NR2B NMDA receptor; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D021122:Protein Subunits; D016194:Receptors, N-Methyl-D-Aspartate