Conformational Selection and Submillisecond Dynamics of the Ligand-binding Domain of the N-Methyl-d-aspartate Receptor

Abstract

The N-methyl-d-aspartate (NMDA) receptors are heteromeric non-selective cation channels that require the binding of glycine and glutamate for gating. Based on crystal structures, the mechanism of partial agonism at the glycine-binding site is thought to be mediated by a shift in the conformational equilibrium between an open clamshell and a closed clamshell-like structure of the bilobed ligand-binding domain (LBD). Using single-molecule Förster resonance energy transfer (smFRET) and multiparameter fluorescence detection, which allows us to study the conformational states and dynamics in the submillisecond time scale, we show that there are at least three conformational states explored by the LBD: the low FRET, medium FRET, and high FRET states. The distance of the medium and low FRET states corresponds to what has been observed in crystallography structures. We show that the high FRET state, which would represent a more closed clamshell conformation than that observed in the crystal structure, is most likely the state initiating activation, as evidenced by the fact that the fraction of the protein in this state correlates well with the extent of activation. Furthermore, full agonist bound LBDs show faster dynamic motions between the medium and high FRET states, whereas they show slower dynamics when bound to weaker agonists or to antagonists.

Computationally Discovered Potentiating Role of Glycans on NMDA Receptors

“…Therefore, any processes on the timescale of ~1–100 μs are unlikely to occur in our simulations of GluN1 and GluN2B LBDs. This work does not attempt to address dynamics on the millisecond and longer timescales414243. Finally, the degree of uncertainty introduced by the use of specific models can be estimated from Table 1, which shows that the timescales of opening/closing in GluN1 and GluN2B LBDs predicted by several models are qualitatively the same.…”

Toward dynamic structural biology: Two decades of single-molecule Förster resonance energy transfer

“…Woźniak et al used time-correlated single photon counting (TCSPC) to explore the bending dynamics of short dsDNA (90). Dolino et al observed submillisecond dynamics in the ligand-binding domain of the N-methyl- d -aspartate receptor (91). Using alternating laser excitation on the nano-second time scale (nsALEX; see Box 1), Laurence et al analyzed fluorescence decays of specific FRET subpopulations to infer an effective distance distribution for the folded and unfolded chemotrypsin inhibitor 2 (CI2) (92).…”

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

“…Using this construct, at least three configurations of the LBD were found. It was suggested that a conformational selection mechanism selectively populated one of the identified populations upon ligand binding 73 . In the inactivated form, or in the presence of the antagonist 5,7-dichlorokynurenic acid (DCKA), mostly medium- to low-FRET states are explored, with a longer donor fluorescence lifetime and a larger donor-to-acceptor fluorescence ratio peaking at F D / F A = 3.3 (Figure 5A).…”

“…It was found that the LBD in the presence of an antagonist shuns the accessibility of a high-FRET state, postulated to be responsible for opening the channel 73 . When comparing experimentally derived distances and the expected values based on crystallographic information, an agreement within 3 Å was achieved.…”