Abstract
The apo state structure of the isolated ligand binding domain of the GluR6 subunit and the conformational changes induced by agonist binding to this protein have been investigated by luminescence resonance energy transfer (LRET) measurements. The LRET-based distances show that agonist binding induces cleft closure, and the extent of cleft closure is proportional to the extent of activation over a wide range of activations, thus establishing that the cleft closure conformational change is one of the mechanisms by which the agonist mediates receptor activation. The LRET distances also provide insight into the apo state structure, for which there is currently no crystal structure available. The distance change between the glutamate-bound state and the apo state is similar to that observed between the glutamate-bound and antagonist UBP-310-bound form of the GluR5 ligand binding domain, indicating that the cleft for the apo state of the GluR6 ligand binding domain should be similar to the UBP-310-bound form of GluR5. This observation implies that te apo state of GluR6 undergoes a cleft closure of 29-30 degrees upon binding full agonists, one of the largest observed in the glutamate receptor family.
Highlights
Methyl-3-hydroxy-4-isoxazole propionate (AMPA)2 subtype, for which currently there are over 60 structures in various ligated states [8, 15, 18]
The few structures of the agonist- and antagonistbound states of the ligand binding domains of the GluR6 and GluR5 subunits of kainate receptors suggest that this subtype of receptors most likely exhibits a similar mechanism as that of the closely related AMPA subtype [13, 14, 17, 18, 33], where the extent of activation is controlled by the extent of cleft closure induced by agonist binding in the ligand binding domain
The structures of the agonist-bound forms of kainate receptors cover a limited range of activations; the only partial agonist structures available are those of the kainate- and domoate-bound states, which exhibit 50% of the efficacy as the full agonist glutamate [13, 14, 18, 33]
Summary
To study the conformational changes in the isolated ligand binding domain of the kainate receptor, we have used a luminescence resonance energy (LRET)-based method to determine distances between sites on domain 1 and domain 2 of the bilobed structure of the GluR6 subunit of the kainate receptor (Fig. 1) in scenarios with a wide range of activations. A wide range of activations was achieved by investigating a number of agonists with the wild type receptor and the T661E mutant. The T661E GluR6 receptors exhibit 20% of the maximum whole cell current by kainate relative to glutamate in the absence of desensitization [34]. These LRET-based distances obtained for the wild type and T661E mutant provide a comprehensive analysis of the changes in cleft closure over the entire spectrum of activations and conclusively show that kainate receptors exhibit a graded cleft closure conformational change and exhibit a similar mechanism of activation as AMPA receptors
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