Conformational changes at the agonist binding domain of the N-methyl-D-aspartic acid receptor

Abstract

The conformational changes in the agonist binding domain of the glycine-binding GluN1 and glutamate-binding GluN2A subunits of the N-methyl D-aspartic acid receptor upon binding agonists of varying efficacy have been investigated by luminescence resonance energy transfer (LRET) measurements. The LRET-based distances indicate a cleft closure conformational change at the GluN1 subunit upon binding agonists; however, no significant changes in the cleft closure are observed between partial and full agonists. This is consistent with the previously reported crystal structures for the isolated agonist binding domain of this receptor. Additionally, the LRET-based distances show that the agonist binding domain of the glutamate-binding GluN2A subunit exhibits a graded cleft closure with the extent of cleft closure being proportional to the extent of activation, indicating that the mechanism of activation in this subunit is similar to that of the glutamate binding α-amino-5-methyl-3-hydroxy-4-isoxazole propionate and kainate subtypes of the ionotropic glutamate receptors.

FIGURE 1.

Sites that were tagged in the (A) GluN1* and (B) GluN2A* subunit to probe for cleft closure conformational changes using LRET measurements are highlighted in the crystal structure of the ABD of GluN1 bound to glycine and GluN2A bound to glutamate.

FIGURE 2.

Cleft closure in the ABD of GluN2A* subunit. A, apo (black)- and antagonist (DLAPV in magenta)-bound LRET lifetimes for GluN1*:GluN2A*N404C-Th, V713C labeled with terbium chelate:fluorescein as measured by the sensitized emission of acceptor at 515 nm. The residuals for the above lifetime fits are shown below each measurement, and the y-axis is in linear scale. B, LRET lifetimes for GluN1*:GluN2A*N404C-Th, V713C labeled with terbium chelate:fluorescein as measured by the sensitized emission of acceptor at 515 nm under saturating concentrations of full agonist (glutamate in black) and partial agonist (HQ in green). The residuals for the above lifetime fits are shown below each measurement the y-axis in linear scale.

FIGURE 3.

Cleft closure in the ABD of GluN1* subunit. LRET lifetimes for GluN1*T396C-Th, A715C:GluN2A* labeled with terbium chelate:ATTO 465 were measured by the sensitized emission of acceptor at 510 nm under saturating concentrations of full agonist (glycine in black) and partial agonists (DCS in green, ACPC in red). The residuals for the above lifetime fits are shown below each measurement, and the y-axis is in linear scale.

FIGURE 4.

Dependence of cleft closure versus extent of activation for the ABD of GluN1* (open squares) and GluN2A* (filled squares) subunit.