N-Terminal Domain of NMDA Receptors as Studied by LRET

Abstract

NMDA Receptors, one of the three main classes of glutamate receptors that mediate excitatory transmission, are tetramers composed of glycine- binding (GluN1) subunits and glutamate- binding (GluN2) subunits. Allosteric regulators are known to bind to the amino-terminal domain of GluN2 subunits; specifically, zinc inhibits GluN2A and GluN2B subunits with varying efficacy. Luminescence resonance energy transfer (LRET) was used to study the soluble construct of the GluN2A and GluN2B amino-terminal domains. Donor and acceptor tags were attached through maleimide linkages to non-disulphide bonded cysteines introduced into lobes 1 and 2 of the bi-lobed amino-terminal domain structure. The LRET based distances of GluN2B are similar to the distances found in the crystal structure indicating that the protein adopts a similar structure in solution as in the crystal structure. Additionally, no significant change in the distance was observed upon addition of zinc, suggesting no significant changes in the average conformation of the bi-lobed protein upon binding zinc. Analogous measurements using the GluN2A amino-terminal domain revealed similar distance measurements to those observed in the GluN2B amino-terminal domain, suggesting a similar conformation of the GluN2A amino-terminal domain to that of the GluN2B amino- terminal domain. Additionally, a small decrease in the distance between the two lobes was observed upon addition of zinc suggesting a possible cleft- closure like conformational change in the GluN2A amino-terminal domain upon addition of saturating concentrations of zinc.