Peptide binding and NMR analysis of the interaction between SAP97 PDZ2 and GluR-A: potential involvement of a disulfide bond.
Synaptic delivery of GluR-A (GluR1) subunit-containing glutamate receptors depends on a C-terminal type I PDZ binding motif in GluR-A. Synapse-associated protein 97 (SAP97) is the only PDZ domain protein known to associate with GluR-A. We have used NMR spectroscopy and a biotinylated peptide binding assay to characterize the interaction between synthetic GluR-A C-terminal peptides and the PDZ2 domain of SAP97 (SAP97(PDZ2)), previously determined to be the dominant factor responsible for the interaction. The binding mode appeared to be strongly influenced by redox conditions. Chemical shift changes observed in NMR spectra indicate that under reducing conditions, the last four residues of GluR-A peptides bind to PDZ2 in a fashion typical of class I PDZ interactions. The binding is weak and relatively nonselective as it occurs similarly with a PDZ2 domain derived from PSD-95, a related protein not believed to directly interact with GluR-A. In the absence of reducing agents, conserved cysteine residues in SAP97(PDZ2) and the GluR-A C-terminus gave rise to an anomalous behavior in a microplate assay with a biotinylated GluR-A 18-mer peptide. A covalent disulfide-linked complex between SAP97(PDZ2) and the GluR-A peptide was seen in the binding assay and in the NMR experiments performed under oxidizing conditions. The results are consistent with a two-step binding mechanism consisting of an initial PDZ interaction followed by stabilization of the complex by a disulfide bond. The possible physiological relevance of redox regulation of SAP97-GluR-A interaction remains to be established.
von Ossowski
Lotta
L
Department of Biological and Environmental Sciences (Division of Biochemistry), University of Helsinki, Helsinki, Finland.
Tossavainen
Helena
H
von Ossowski
Ingemar
I
Cai
Chunlin
C
Aitio
Olli
O
Fredriksson
Kai
K
Permi
Perttu
P
Annila
Arto
A
Keinänen
Kari
K
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019921:Biotinylation; D002247:Carbon Isotopes; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D011485:Protein Binding / Q000502:physiology*; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D051381:Rats; D018091:Receptors, AMPA / Q000737:chemistry / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D002247:Carbon Isotopes; D004220:Disulfides; D008565:Membrane Proteins; D018091:Receptors, AMPA; C501119:Dlg1 protein, rat