Identification of an atypical calcium-dependent calmodulin binding site on the C-terminal domain of GluN2A
| Autor: | Philip R. Gafken, Andrew M. Hau, Jane E. Ishmael, Michael I. Schimerlik, Gaurav Bajaj, Peter L. Hsu |
|---|---|
| Rok vydání: | 2014 |
| Předmět: |
Calmodulin
Protein subunit Molecular Sequence Data Biophysics Glutamic Acid Plasma protein binding Biochemistry Receptors N-Methyl-D-Aspartate Article Mice Animals Amino Acid Sequence Binding site Molecular Biology Peptide sequence Ion channel Binding Sites biology C-terminus Cell Biology Protein Structure Tertiary Rats biology.protein Calcium Sequence motif Protein Binding |
| Zdroj: | Biochemical and biophysical research communications. 444(4) |
| ISSN: | 1090-2104 |
| Popis: | N-methyl- d -aspartate (NMDA) receptors are calcium-permeable ion channels assembled from four subunits that each have a common membrane topology. The intracellular carboxyl terminal domain (CTD) of each subunit varies in length, is least conserved between subunits, and binds multiple intracellular proteins. We defined a region of interest in the GluN2A CTD, downstream of well-characterized membrane-proximal motifs, that shares only 29% sequence similarity with the equivalent region of GluN2B. GluN2A (amino acids 875–1029) was fused to GST and used as a bait to identify proteins from mouse brain with the potential to bind GluN2A as a function of calcium. Using mass spectrometry we identified calmodulin as a calcium-dependent GluN2A binding partner. Equilibrium fluorescence spectroscopy experiments indicate that Ca2+/calmodulin binds GluN2A with high affinity (5.2 ± 2.4 nM) in vitro. Direct interaction of Ca2+/calmodulin with GluN2A was not affected by disruption of classic sequence motifs associated with Ca2+/calmodulin target recognition, but was critically dependent upon Trp-1014. These findings provide new insight into the potential of Ca2+/calmodulin, previously considered a GluN1-binding partner, to influence NMDA receptors by direct association. |
| Databáze: | OpenAIRE |
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