von Ossowski Lotta, Tossavainen Helena, von Ossowski Ingemar, Cai Chunlin, Aitio Olli, Fredriksson Kai, Permi Perttu, Annila Arto, Keinänen Kari
Department of Biological and Environmental Sciences (Division of Biochemistry), University of Helsinki, Helsinki, Finland.
Biochemistry. 2006 May 2;45(17):5567-75. doi: 10.1021/bi0511989.
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.
含GluR - A（GluR1）亚基的谷氨酸受体的突触传递依赖于GluR - A中C末端I型PDZ结合基序。突触相关蛋白97（SAP97）是已知与GluR - A相关的唯一PDZ结构域蛋白。我们使用核磁共振光谱和生物素化肽结合试验来表征合成的GluR - A C末端肽与SAP97的PDZ2结构域（SAP97(PDZ2)）之间的相互作用，先前已确定该结构域是负责这种相互作用的主要因素。结合模式似乎受到氧化还原条件的强烈影响。核磁共振光谱中观察到的化学位移变化表明，在还原条件下，GluR - A肽的最后四个残基以I类PDZ相互作用的典型方式与PDZ2结合。这种结合较弱且相对非选择性，因为它与源自PSD - 95的PDZ2结构域（一种不被认为直接与GluR - A相互作用的相关蛋白）的结合情况类似。在没有还原剂的情况下，SAP97(PDZ2)和GluR - A C末端中的保守半胱氨酸残基在使用生物素化的GluR - A 18聚体肽的微孔板试验中产生了异常行为。在结合试验和氧化条件下进行的核磁共振实验中都观察到了SAP97(PDZ2)与GluR - A肽之间的共价二硫键连接复合物。结果与两步结合机制一致，该机制包括初始的PDZ相互作用，随后通过二硫键稳定复合物。SAP97 - GluR - A相互作用的氧化还原调节的可能生理相关性仍有待确定。
