Crystal structures for the isolated ATDs of the NMDA receptor GluN1 and GluN2 subunits are especially interesting since they reveal monomer, dimer and tetramer assemblies with strikingly different conformations from those for AMPA and kainate receptors, suggesting that the global architecture of subunit packing in NMDA receptor tetramers might differ from that for AMPA and kainate receptors (). Although dimer assemblies of the NMDA receptor LBDs closely resemble those for AMPA and kainate receptors (Furukawa et al., 2005
), the three NMDA receptor ATD structures solved to date, for rat and Xenopus
GluN1 and for rat GluN2B, all exhibit twisted conformations in which the relative orientation of the lower lobe is rotated by approximately 45° compared to the conformation found in AMPA and kainate receptors (Farina et al., 2011
; Karakas et al., 2009
; Karakas et al., 2011
). The rat and Xenopus
GluN1 ATD structures are essentially identical (rmsd 0.99 Å) with the exception that the rat construct lacks 21 residues encoded by exon 5, which were present in the Xenopus
GluN1 splice variant used for crystallization. Like the battery of recently solved AMPA and kainate receptor structures (), the GluN1 ATD was crystallized in the apo state, while the GluN2B ATD was crystallized in a complex with Zn2+
as well as in the apo state. In addition, a GluN2B ifenprodil complex was solved for a GluN1/GluN2B ATD heterodimer. It is remarkable that all three GluN2B structures have essentially identical, closed cleft conformations, with rmsds of 0.52 and 1.0 Å for superposition on the GluN2B apo structure, which is turn is similar to the apo structures of the GluN1 ATDs. There is evidence from functional studies, which used either Cys mutant cross linking (Stroebel et al., 2011
), or chemical modification by MTS reagents with bulky substituents (Gielen et al., 2009
), that in their apo state the GluN2A and GluN2B ATDs must be able to adopt an open cleft conformation, but to date this conformation has not been crystallized, and the extent of the conformational change underlying the open to closed transition remains to be established. It is also unknown whether the GluN1 ATD can adopt an open cleft conformation, or whether like the AMPA and kainate receptor ATDs it remains closed in the absence of ligands.
NMDA receptor ATD dimer and tetramer structures
An even more remarkable difference for NMDA and non-NMDA receptor ATDs emerges on inspection of crystal structures for their dimer and tetramer assemblies, but this raises an important caveat. The ATD structures were solved for isolated domains expressed as soluble proteins removed from restraints present in a tetrameric membrane protein assembly. For AMPA and kainate receptors the similar structure of the isolated ATD and LBD dimer and tetramer assemblies to those found in the full length GluA2 structure leaves little doubt about their biological significance. However, there are a handful of LBD dimer assemblies in the PBD which likely have no biological significance. Some of these ‘fakes’ are easy to spot, for example dimers in which the subunits are rotated by 180°, to generate head to tail assemblies, but for others the changes are more subtle, and can be eliminated only by considering sources of additional information gained for example from functional experiments and site directed mutagenesis. The highly flexible nature of an iGluR tetramer assembly, for which the LBDs undergo large conformational changes during the processes of activation and desensitization, adds to the complexity, since multiple conformational states of the ATD and LBD tetramer assemblies must occur, but at present we have little information on what these should look like. Further difficulties arise if the GluA2 structure cannot be used as a template for NMDA receptors.
With these caveats in mind, the NMDA receptor ATD structures are fascinating in their own right. In the three crystallographically independent GluN1 ATD dimers, one from Xenopus, and two from rat, intermolecular contacts are mediated exclusively via the upper lobes of the ATD clam shell, with a buried surface of 1140 Å per subunit. Due to the 45° twist of the lower lobes in each protomer, away from the axis of dimer symmetry, the lower lobe plays no role in dimer assembly. Different from the ATD dimer assemblies for AMPA and kainate receptors, where α-helix B from one subunit faces αhelixC of its dimer partner, in the GluN1 homodimer the subunits are displaced laterally, with a 90° rotation of one subunit compared to the assembly of AMPA and kainate receptor ATDs. As a consequence, in the GluN1 homodimer α-helix C is in contact with α-helix C in its dimer partner, while α-helix B is located at the lateral edge of the dimer assembly. Superposition of the GluN1/GluN2B heterodimer on the GluN1 homodimer, using coordinates for one of the GluN1 protomers, reveals a strikingly different dimer assembly, in which the GluN2B subunit is rotated by 180° compared to the GluN1 homodimer (). Despite this, the buried surface in the GluN1/GluN2B heterodimer, 1100 to 1200 Å per subunit, is similar in size to that in the GluN1 homodimer, although the contacts are mediated by different sets of interactions between α-helices B andC. In addition, for the GluN1/GluN2B heterodimer, a loop connecting β-strand 6 to α-helix G in the lower lobe of the GluN2B subunit projects into the dimer interface and makes contacts with the upper lobe of the GluN1 subunit. Overall, a superposition using coordinates for the upper lobes of the ATDs reveals that the GluN1/GluN2B heterodimer assembly more closely resembles that found in AMPA and kainate receptors than the GluN1 homodimer.
The GluN1/GluN2B ATD crystal structure contains 2 dimer pairs in the asymmetric unit, and these form a tetramer with the ‘dimer of dimers’ interface mediated by the GluN2 subunits (), consistent with Cys mutant cross linking experiments on the LBD and the predicted tetrameric organization of an NMDA receptor (Sobolevsky et al., 2009
). However the dimer of dimers interface is completely different from that found in AMPA and kainate receptors, is formed by the upper not lower lobes of the ATD protomers, and involves contacts mediated by the S-loop. Without additional information it is impossible to tell if the GluN1/GluN2B NCS tetramer corresponds to a native NMDA receptor ATD conformation, or occurs only in the crystal lattice. Perhaps wisely, Karakas et al., (2011)
chose not to discuss the GluN1/GluN2B tetramer assembly, and focused on the dimer assembly and its role in allosteric modulation.
Gel filtration and sedimentation analysis reveals that the isolated ATDs for GluN1 and GluN2 are monomeric at protein concentrations above 1 mg/ml, but form high affinity heterodimers when mixed (Karakas et al., 2011
). Thus the functional significance of GluN1 ATD homodimer crystal structures could be questionable. It is striking however, that the rat and Xenopus
structures are nearly identical; that they were solved in different space groups (rat P31
); and that in the Xenopus
structure there are two identical dimers generated by crystallographic and non crystallographic symmetry; thus, despite low affinity for self association, the GluN1 ATDs appear to have a low energy state favoring dimer assembly in solution, as also supported by single particle EM analysis (Farina et al., 2011
). Based on these observations, it has been proposed that the formation of NR1 homodimers occurs as an assembly intermediate prior to exchange with GluN2 subunits (Farina et al., 2011
); similar proposals have been made for biosynthesis of heteromeric AMPA receptors, during which GluA2 is likely to form high affinity homodimer assemblies (Rossmann et al., 2011
). Dimer dissociation, and subunit exchange would likely require mechanisms that stabilize the fold of individual iGluR subunits before assembly as a tetramer. Possibly the exchange could occur before the ion channel segments are inserted into the lipid bilayer, or perhaps there are novel membrane proteins which act as chaperones and stabilize lipid embedded iGluR monomers and dimers. Very little is known about these early stages of ion channel assembly.