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J. Biol. Chem., Vol. 280, Issue 29, 26992-26996, July 22, 2005

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Structural Characterization of the Intermolecular Interactions of Synapse-associated Protein-97 with the NR2B Subunit of N-Methyl-D-aspartate Receptors^*

Lei Wang, Andrea Piserchio, and Dale F. Mierke¶

From the Department of Physics and Department of Molecular Pharmacology, Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912

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 2/3 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 2/3 loop provides an avenue for regulating the ligand specificity of PDZ domains.

Received for publication, March 31, 2005 , and in revised form, May 18, 2005.

^* This work was supported by Grants GM-54082 and DA-18428 from the National Institute of Health (to D. F. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Molecular Pharmacology, Division of Biology and Medicine, Box G-B4, Brown University, Providence, RI 02912. Tel.: 401-863-2139; Fax: 401-863-1595; E-mail: dale_mierke{at}brown.edu.

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