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Expression and characterization of soluble amino-terminal domain of NR2B subunit of N-methyl-D-aspartate receptor

¹ Glutamate Receptor Laboratory, National Neuroscience Institute, S308433, Singapore
² Department of Pharmacology, National University of Singapore, S117597, Singapore
³ Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia 30322-3090, USA

(RECEIVED April 11, 2005; FINAL REVISION May 30, 2005; ACCEPTED May 30, 2005)

N-methyl-D-aspartate (NMDA) receptors are involved in mediating excitatory synaptic transmissions in the brain and have been implicated in numerous neurologic disorders. The proximal amino-terminal domains (ATDs) of NMDA receptors constitute many modulatory binding sites that may serve as potential drug targets. There are few biochemical and structural data on the ATDs of NMDA receptors, as it is difficult to produce the functional proteins. Here an optimized method was established to reconstitute the insoluble recombinant ATD of NMDA receptor NR2B subunit (ATD2B) through productive refolding of 6xHis-ATD2B protein from inclusion bodies. Circular dichroism and dynamic light scattering characterizations revealed that the solubilized and refolded 6xHis-ATD2B adopted well-defined secondary structures and monodispersity.More significantly, the soluble 6xHis-ATD2B specifically bound ifenprodil to saturation. Ifenprodil bound to 6xHis-ATD2B with a dissociation constant (K_D) of 127.5±45 nM, which was within the range of the IC₅₀ determined electrophysiologically. This is the first report on a functional recombinant ATD2B with a characterized K_D.

Keywords: NR2B NMDA receptor; protein refolding; circular dichroism; dissociation constant; dynamic light scattering

Abbreviations: NMDA, N-methyl-D-aspartate • ATD, amino-terminal domain • CD, circular dichroism • DLS, dynamic light scattering • K_D, dissociation constant • LIVBP, leucine/isoleucine/valine-binding protein • MALDI-TOF, matrix-assisted laser desorption ionization time of flight • MS, mass spectrometry

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051509905.

Reprint requests to: Chian-Ming Low, Department of Pharmacology, MD2 #04-22, Faculty of Medicine, National University of Singapore, 18 Medical Drive, S117597, Singapore; e-mail: phclowcm{at}nus.edu.sg; fax: +(65) 68737690.

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