Structure, Dynamics, and Allosteric Potential of Ionotropic Glutamate Receptor N-Terminal Domains

Abstract

Ionotropic glutamate receptors (iGluRs) are tetrameric cation channels that mediate synaptic transmission and plasticity. They have a unique modular architecture with four domains: the intracellular C-terminal domain (CTD) that is involved in synaptic targeting, the transmembrane domain (TMD) that forms the ion channel, the membrane-proximal ligand-binding domain (LBD) that binds agonists such as L-glutamate, and the distal N-terminal domain (NTD), whose function is the least clear. The extracellular portion, comprised of the LBD and NTD, is loosely arranged, mediating complex allosteric regulation and providing a rich target for drug development. Here, we briefly review recent work on iGluR NTD structure and dynamics, and further explore the allosteric potential for the NTD in AMPA-type iGluRs using coarse-grained simulations. We also investigate mechanisms underlying the established NTD allostery in NMDA-type iGluRs, as well as the fold-related metabotropic glutamate and GABAB receptors. We show that the clamshell motions intrinsically favored by the NTD bilobate fold are coupled to dimeric and higher-order rearrangements that impact the iGluR LBD and ultimately the TMD. Finally, we explore the dynamics of intact iGluRs and describe how it might affect receptor operation in a synaptic environment.

Figure 1

Structure of iGluR NTDs. (A) A structure of a homomeric GluA2 AMPAR (PDB ID: 3KG2) (18) is shown with the three main domain layers colored red (NTD), yellow (LBD), and green (TMD). The interdomain linkers are colored black. One subunit is highlighted. (B) A zoom-in of an NTD dimer illustrates the clamshell structure of each protomer (upper lobe (UL) and lower lobe (LL) separated by a cleft), as well as four features that vary between iGluRs: the overall dimeric packing, the top loop, the wing element, and the side loop. (C) The dimer interfaces of selected iGluR NTD dimers are shown as color-coded spheres for atoms forming contacts (interfacial atom-to-atom distance = <4.5 Å). Spheres are colored according to the number of contacts, from blue (n = 1) to red (n ≥ 7). A spectrum of LL packing is shown, comparing iGluR NTD dimers (left to right). The kainate receptors (exemplified by a GluK2 homodimer; PDB ID: 3H6H) (60) show the most LL packing similar to their UL packing (these interfaces show local contact densities (LDs) (100) of 27.0 and 35.1, respectively). Among the AMPAR paralogs, homodimeric LL packing correlates with affinity with GluA2 (PDB ID: 3H5V, dimer AB) (46) having the most tightly packed LLs (LD 23.4 vs. 43.5 for UL) and GluA3 (PDB ID: 3O21, dimer CD) (57) having minimal LL packing (LD 6.7 vs. 45.0 for UL). The NMDARs are at the far end of the spectrum, with no LL packing at all (shown for GluN1 of the heterodimer; PDB ID: 3QEL, dimer AB) (64). The dashed red circles in GluA3 and GluN1 highlight the lack of LL packing. A more detailed analysis of NTD dimer interfaces can be found in our recent studies (32, 56, 57). To see this figure in color, go online.

Figure 2

Motions accessible to iGluR NTD monomers. (A) Mode 1 is an interlobe twist. This is illustrated for AMPAR paralog GluA3 (PDB ID: 3O21, chain C) (57) using a front view based on the ULs (approximately constant position). The dimer interface is colored red (UL) and pink (LL). An arbitrary extent of motion is shown. (B) Mode 2 is a classical PBP cleft motion. A side view is shown with the cleft angle indicated by three marker residues (one in the UL, one in the hinge region, and one in the LL). Again the dimer interface is colored red and pink, and the extent of motion is arbitrary. (C and D) The extents of motion (square displacements) for the twisting motion (C) and cleft closure motion (D) are compared across iGluR NTDs (NMDARs in red) and the NTD-like allosteric modules from subunits of metabotropic glutamate (mGlu) and GABA_B (GB) receptors (both type C GPCRs; blue). The scale is relative to the twisting motion of an open mGlu1 clamshell (PDB ID: 1EWV) (30). The twisting motion (C) shows two clear clusters besides the open mGlu1, highlighted in blue and yellow. This segregation is less clear for the cleft motion (D). More details of the structures used for this analysis are listed in Table S1. (E) The ANM modes of the GB2 NTD-like module are compared against the conformational transition induced upon activation of the dimer by GABA binding to the GB1 subunit. Correlation cosines (or overlaps) are shown as blue bars. The red curve shows the cumulative overlap (root mean square of the correlation cosines). A twisting motion stands out (the asterisk marks mode 1). (F) The twisting motion (the asterisk marks mode 1) also stands out when we assess the overlaps between the ANM modes of the GluA3 NTD monomer and the difference between the structures of GluA3 and GluN2B NTD monomers. (G) A comparison of the ANM modes of the GB1 module against its conformational change upon GABA binding reveals a cleft closure motion (the asterisk marks mode 2).

Figure 3

Motions that are accessible to dimers. (A) mGluRs have been captured in a displaced resting (R) state (exemplified by PDB ID: 1EWT) (30) and a parallel active (A) state (stabilized by glutamate and cations, green and orange spheres; PDB ID: 1ISR) (82). Front and side views are shown. (B) These structures can interconvert with mode 1 (an interprotomer counterrotation), accounting for most of the transition. The blue bars and red curve are overlaps and cumulative overlaps, respectively, as in Fig. 2. (C) The extent of this motion is compared across NTDs relative to the monomer motions (mGlu1 twisting = 1). The GluA3 AMPAR NTD homodimer stands out as a result of its decreased LL packing (similar to state A mGlu1, yellow). More details of the structures used for this analysis are listed in Table S1. (D) Right: GluA3 was captured in a conformation resembling the mGluR A state. Left: ANM mode 1 enables the GluA3 NTD dimer to reach a conformation resembling the R state. (E) NTD dimers also exhibit cleft motions in the ANM (56) and in all-atom MD simulations. Variations in the Cα distance between A47 and D193 in simulations of a GluA2 NTD dimer (PDB ID: 3H5V) (46) reveal an interlobe twist similar to that of monomer ANM Mode 1. The first 25 ns portion of one simulation is shown for this distance in one subunit. The horizontal stippled line denotes the 17.5 Å Cα distance seen in the crystal structure. The GluA2 NTD structure on the right shows the position of the marker residues (A47, blue; D193, red) and the distance between their Cα residues (stippled line between dark gray spheres).

Figure 4

(A and B) Monomer and dimer conformational changes are coupled to motions of NTD tetramers and whole receptors in both NMDARs (A) and AMPARs (B). Matrices comparing the modes of motion of these smaller systems with progressively larger systems are represented as colored squares. Darker colors indicate higher correlations. Red (positive correlations) and blue (negative correlations) are equivalent, as ANM modes are harmonic fluctuations with arbitrary starting directions. The bottom matrix (panel 1) shows the overlap between modes of monomer motions (ordinate, label on far right and shared by AMPARs and NMDARs) and dimer motions (abscissa). A zoom into the first few elements illustrates that many dimer modes show a significant overlap with both prominent modes of monomer motion. The matrix above (panel 2) compares the same dimer modes of motion (shared abscissa) with the modes of motion of the NTD tetramer (ordinate, label on far right). In this case, there are many darker blocks, indicating a higher correlation. The top-left matrix shows the overlap between the motions of the NTD tetramer and those of the whole receptor. The starting structure for the AMPAR is a GluA2 homotetramer (18); the NMDAR is a GluN1/2B heteromer (PDB ID: 4PE5) (16). Lines connect some modes of motion that show good correlations all the way from monomers to whole receptors. Dimeric motions are more conserved through the levels in the NMDAR, as illustrated for dimer mode 1, where dark dots illustrate good correlations along the way and the endpoints are the circled modes 6–8 of the whole NMDAR. The monomer shown is GluN1, where cleft motions are also well retained. In the AMPAR monomer, cleft motions are again retained but dimer rearrangements are dampened in the whole receptor. This results in poor correlations transitioning from the NTD tetramer to the whole receptor (white dots and stippled trapezium). Also evident is a higher dominance of tetramer motions in lower-frequency modes of the NMDAR, where the tighter ECR packing restricts rearrangements in which the NTD moves as a rigid body.

Figure 5

ANM of the whole GluA2 AMPAR shows global bending motions that could bring the NTD into proximity with auxiliary subunits such as TARPs. An AMPAR crystal structure (PDB ID: 3KG2) (18) is shown on the left, with the two nonequivalent chain pairs colored in red (distal from the NTD tetramer interface) and gray (proximal and interface forming). A homology model of TARP γ-2 based on the related claudins is shown in cyan. The structure on the right shows a conformation along ANM mode 1 in which the NTD bends down and can contact the TARP. To see this figure in color, go online.