The loss of an electrostatic contact unique to AMPA receptor ligand binding domain 2 slows channel activation

Ligand-gated ion channels undergo conformational changes that transfer the energy of agonist binding to channel opening. Within ionotropic glutamate receptor (iGluR) subunits, this process is initiated in their bilobate ligand binding domain (LBD) where agonist binding to lobe 1 favors closure of lobe 2 around the agonist and allows formation of interlobe hydrogen bonds. AMPA receptors (GluAs) differ from other iGluRs because glutamate binding causes an aspartate-serine peptide bond in a flexible part of lobe 2 to rotate 180° (flipped conformation), allowing these residues to form cross-cleft H-bonds with tyrosine and glycine in lobe 1. This aspartate also contacts the side chain of a lysine residue in the hydrophobic core of lobe 2 by a salt bridge. We investigated how the peptide flip and electrostatic contact (D655-K660) in GluA3 contribute to receptor function by examining pharmacological and structural properties with an antagonist (CNQX), a partial agonist (kainate), and two full agonists (glutamate and quisqualate) in the wildtype and two mutant receptors. Alanine substitution decreased the agonist potency of GluA3(i)-D655A and GluA3(i)-K660A receptor channels expressed in HEK293 cells and differentially affected agonist binding affinity for isolated LBDs without changing CNQX affinity. Correlations observed in the crystal structures of the mutant LBDs included the loss of the D655-K660 electrostatic contact, agonist-dependent differences in lobe 1 and lobe 2 closure, and unflipped D(A)655-S656 bonds. Glutamate-stimulated activation was slower for both mutants, suggesting that efficient energy transfer of agonist binding within the LBD of AMPA receptors requires an intact tether between the flexible peptide flip domain and the rigid hydrophobic core of lobe 2.     

Population Shift Mechanism for Partial Agonism of AMPA Receptor

α-amino-3-hydroxy-5-methyl-4-isoaxazolepropionic acid (AMPA) ionotropic glutamate receptors mediate fast excitatory neurotransmission in the central nervous system, and their dysfunction is associated with neurological diseases. Glutamate binding to ligand-binding domains (LBDs) of AMPA receptors induces channel opening in the transmembrane domains of the receptors. The T686A mutation reduces glutamate efficacy so that the glutamate behaves as a partial agonist. The crystal structures of wild-type and mutant LBDs are very similar and cannot account for the observed behavior. To elucidate the molecular mechanism inducing partial agonism of the T686A mutant, we computed the free-energy landscapes governing GluA2 LBD closure using replica-exchange umbrella sampling simulations. A semiclosed state, not observed in crystal structures, appears in the mutant during simulation. In this state, the LBD cleft opens slightly because of breaking of interlobe hydrogen bonds, reducing the efficiency of channel opening. The energy difference between the LBD closed and semiclosed states is small, and transitions between the two states would occur by thermal fluctuations. Evidently, glutamate binding to the T686A mutant induces a population shift from a closed to a semiclosed state, explaining the partial agonism in the AMPA receptor.     

Glutamate Binding and Conformational Flexibility of Ligand-binding Domains Are Critical Early Determinants of Efficient Kainate Receptor Biogenesis

Intracellular glutamate binding within the endoplasmic reticulum (ER) is thought to be necessary for plasma membrane expression of ionotropic glutamate receptors. Here we determined the importance of glutamate binding to folding and assembly of soluble ligand-binding domains (LBDs), as well as full-length receptors, by comparing the secretion of a soluble GluR6-S1S2 protein versus the plasma membrane localization of GluR6 kainate receptors following mutagenesis of the LBD. The mutations were designed to either eliminate glutamate binding, thereby trapping the bilobate LBD in an “open” conformation, or “lock” the LBD in a closed conformation with an engineered interdomain disulfide bridge. Analysis of plasma membrane localization, medium secretion of soluble LBD proteins, and measures of folding efficiency suggested that loss of glutamate binding affinity significantly impacted subunit protein folding and assembly. In contrast, receptors with conformationally restricted LBDs also exhibited decreased PM expression and altered oligomeric receptor assembly but did not exhibit any deficits in subunit folding. Secretion of the closed LBD protein was enhanced compared with wild-type GluR6-S1S2. Our results suggest that glutamate acts as a chaperone molecule for appropriate folding of nascent receptors and that relaxation of LBDs from fully closed states during oligomerization represents a critical transition that necessarily engages other determinants within receptor dimers. Glutamate receptor LBDs therefore must access multiple conformations for efficient biogenesis.Cellular control over the biogenesis and trafficking of ionotropic glutamate receptors (iGluRs)² probably constitutes a major regulatory process that prevents aberrant excitatory neurotransmission within the central nervous system. Many receptor trafficking determinants are genetically encoded within the primary amino acid sequences of cytoplasmic carboxyl-terminal domains of iGluR subunits (1, 2). Association of these determinants with accessory or chaperone proteins enhances forward transit from the endoplasmic reticulum (ER), insertion into the plasma membrane (PM), synaptic targeting, or a variety of other trafficking processes (3–6). Ligand binding and gating domains upstream of carboxyl-terminal domains also are critical in biogenesis of iGluRs (7–11). Elucidating how these domains are engaged as quality control checkpoints is essential to developing a comprehensive understanding of the neuronal mechanisms for control of excitatory transmission in the central nervous system.Several recent studies have proposed that intracellular glutamate binding within the ER promotes proper folding and maturation of AMPA and kainate receptors and is required for forward trafficking of fully assembled receptors (10, 12–15). This hypothesis is based in large part on the observation that mutagenesis of key glutamate-binding residues in the LBD greatly reduces or eliminates PM localization (7, 10, 12, 13); the mechanistic basis of this putative quality control process is not understood. The critical receptor determinants involved in these cellular checkpoints could reside in the LBD, in other regions that undergo glutamate-dependent alterations in structure, or in intersubunit interactions associated with receptor desensitization (7–10, 16). It is possible that glutamate binding to iGluRs (and subsequent conformational rearrangements) act as tests of the functionality of fully assembled, export-ready receptors (8, 12, 16); alternatively, intact glutamate-binding sites may be required for earlier steps in receptor biogenesis, such as subunit folding and oligomeric assembly (7, 16).We tested here whether the glutamate-dependent conformational changes critical to GluR6a KAR cell surface expression are restricted solely to the bilobate LBD, formed by the S1 and S2 segments of the receptor protein, or instead involve additional domains in the KAR subunits. Toward that end, we compared the effect of an LBD mutation that eliminated binding affinity for glutamate, T690A, on the efficiency of secretion of a soluble GluR6-S1S2 protein versus PM localization of the full-length receptor; this served as one way to test the autonomy of the LBD in quality control processes, because soluble LBDs fold properly, form binding sites similar to those in the full-length receptor, and transit the secretory pathway to be released into culture media. Furthermore, we trapped LBDs of full-length receptors and soluble GluR6-S1S2 proteins in conformations that mimic closed, glutamate-bound states by introduction of a reversible, interdomain (D1-D2) disulfide bond, allowing us to test the role of LBD relaxation or flexibility in assembly and trafficking. We found that the ligand binding mutation resulted in reduced maturation and receptor surface expression in part through misfolding of the LBD. In addition, we found that locking the LBD into a closed state reduced receptor maturation and expression but that this engaged regions outside of the LBD. Although the locked, closed mutant also had assembly defects that occurred at the transition from dimers to tetramers, these did not result from apparent folding defects, suggesting that relaxation or opening of the LBD represents a critical step during oligomerization. In summary, we propose that LBDs must be able to access multiple conformations for efficient KAR biogenesis.     

Kainate induces various domain closures in AMPA and kainate receptors

Ionotropic glutamate receptors are key players in fast excitatory synaptic transmission within the central nervous system. These receptors have been divided into three subfamilies: the N-methyl-d-aspartic acid (NMDA), 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) and kainate receptors. Kainate has previously been crystallized with the ligand binding domain (LBD) of AMPA receptors (GluA2 and GluA4) and kainate receptors (GluK1 and GluK2). Here, we report the structures of the kainate receptor GluK3 LBD in complex with kainate and GluK1 LBD in complex with kainate in the absence of glycerol. Kainate introduces a conformational change in GluK3 LBD comparable to that of GluK2, but different from the conformational changes induced in GluA2 and GluK1. Compared to their domain closures in a glutamate bound state, GluA2 and GluK1 become more open and kainate induces a domain closure of 60% and 62%, respectively, relative to glutamate (100%). In GluK2 and GluK3 with kainate, the domain closure is 88% and 83%, respectively. In previously determined structures of GluK1 LBD in complex with kainate, glycerol is present in the binding site where it bridges interlobe residues and thus, might contribute to the large domain opening. However, the structure of GluK1 LBD with kainate in the absence of glycerol confirms that the observed domain closure is not an artifact of crystallization conditions. Comparison of the LBD structures with glutamate and kainate reveals that contacts are lost upon binding of kainate in the three kainate receptors, which is in contrast to the AMPA receptors where similar contacts are seen. It was revealed by patch clamp electrophysiology studies that kainate is a partial agonist at GluK1 with 36% efficacy compared to glutamate, which is in between the published efficacies of kainate at GluK2 and AMPA receptors. The ranking of efficacies seems to correlate with LBD domain closures.     

Correlating efficacy and desensitization with GluK2 ligand-binding domain movements

Gating of AMPA- and kainate-selective ionotropic glutamate receptors can be defined in terms of ligand affinity, efficacy and the rate and extent of desensitization. Crucial insights into all three elements have come from structural studies of the ligand-binding domain (LBD). In particular, binding-cleft closure is associated with efficacy, whereas dissociation of the dimer formed by neighbouring LBDs is linked with desensitization. We have explored these relationships in the kainate-selective subunit GluK2 by studying the effects of mutating two residues (K531 and R775) that form key contacts within the LBD dimer interface, but whose truncation unexpectedly attenuates desensitization. One mutation (K531A) also switches the relative efficacies of glutamate and kainate. LBD crystal structures incorporating these mutations revealed several conformational changes that together explain their phenotypes. K531 truncation results in new dimer contacts, consistent with slower desensitization and sideways movement in the ligand-binding cleft correlating with efficacy. The tested mutants also disrupted anion binding; no chloride was detected in the dimer-interface site, including in R775A where absence of chloride was the only structural change evident. From this, we propose that the charge balance in the GluK2 LBD dimer interface maintains a degree of instability, necessary for rapid and complete desensitization.     

Structural basis for retinoic X receptor repression on the tetramer

Retinoic X receptor (RXR) is a master nuclear receptor in the processes of cell development and homeostasis. Unliganded RXR exists in an autorepressed tetramer, and agonists can induce RXR dimerization and coactivator recruitment for activation. However, the molecular mechanisms involving the corepressor recruitment and antagonist-mediated repression of RXR are still elusive. Here we report the crystal structure of RXRα ligand-binding domain (LBD) complexed with silencing mediator for retinoid and thyroid hormone receptors (SMRT) corepressor motif. As the first structural report on the unliganded nuclear receptor bound to the corepressor motif, RXRαLBD-SMRT exhibits a significant structural rearrangement, compared with apoRXRαLBD tetramer. To elucidate further the molecular determinants for RXR repression by its antagonist, we also determine the crystal structure of RXRαLBD-SMRT complexed with the identified antagonist rhein. In the structure, two rhein molecules and two SMRT peptides are in the RXRαLBD tetramer, different from the case in RXRαLBD-SMRT structure, where four SMRT peptides bind to RXRαLBD tetramer. It seems that rhein induces a displacement of SMRT motif by activation function 2 (AF-2) motif binding to the receptor. Combining our current work with the published results, structural superposition of RXRαLBDs in different states reveals that RXR uses an overlapped binding site for coactivator, corepressor, and AF-2 motifs, whereas the AF-2 motif adopts different conformations for agonist or antagonist interaction and coactivator or corepressor recruitment. Taken together, we thus propose a molecular model of RXR repression on the tetramer.     

Low-resolution structures of thyroid hormone receptor dimers and tetramers in solution

High-resolution X-ray structures of thyroid hormone (TH) receptor (TR) DNA and ligand binding domains (DBD and LBD) have yielded significant insights into TR action. Nevertheless, the TR DBD and LBD act in concert to mediate TH effects upon gene expression, and TRs form multiple oligomers; however, structures of full-length TRs or DBD-LBD constructs that would clarify these influences are not available. Here, we report low-resolution X-ray structures of the TRbeta DBD-LBD construct in solution which define the shape of dimers and tetramers and likely positions of the DBDs and LBDs. The holo TRbeta DBD-LBD construct forms a homodimer with LBD-DBD pairs in close contact and DBDs protruding from the base in the same direction. The DBDs are connected to the LBDs by crossed extended D domains. The apo hTRbeta DBD-LBD construct forms tetramers that resemble bulged cylinders with pairs of LBD dimers in a head-to-head arrangement with DBD pairs packed tightly against the LBD core. Overall, there are similarities with our previous low-resolution structures of retinoid X receptors, but TRs exhibit two unique features. First, TR DBDs are closely juxtaposed in the dimer and tetramer forms. Second, TR DBDs are closely packed against LBDs in the tetramer, but not the dimer. These findings suggest that TRs may be able to engage in hitherto unknown interdomain interactions and that the D domain must rearrange in different oligomeric forms. Finally, the data corroborate our suggestion that apo TRs form tetramers in solution which dissociate into dimers upon hormone binding.     

The action of Con-ikot-ikot toxin on single AMPA-type glutamate receptors

Conotoxins are a large group of naturally occurring toxic peptides produced by the predatory sea snails of the genus Conus. Many of these toxins target ion channels, often with high specificity and affinity. As such, they have proven to be invaluable for basic research, as well as acting as leads for therapeutic strategies. Con-ikot-ikot is the only conotoxin so far identified that targets AMPA-type glutamate receptors, the main mediators of excitatory neurotransmission in the vertebrate brain. Here, we describe how the toxin modifies the activity of AMPA receptors at the single-channel level. The toxin binds to the AMPA receptor with EC₅₀ of 5 nM, and once bound takes minutes to wash out. As shown previously, it effectively blocks desensitization of AMPA receptors; however, compared to other desensitization blockers, it is a poor stabilizer of the open channel because toxin-bound AMPA receptors undergo frequent brief closures. We propose that this is a direct consequence of the toxin’s unique binding mode to the ligand-binding domains (LBDs). Unlike other blockers of desensitization, which stabilize individual dimers within an AMPA receptor tetramer, the toxin immobilizes all four LBDs of the tetramer. This result further emphasizes that quaternary reorganization of independent LBD dimers is essential for the full activity of AMPA receptors.     

Assembly and Trafficking of Homomeric and Heteromeric Kainate Receptors with Impaired Ligand Binding Sites

Kainate receptors (KARs) are a subfamily of ionotropic glutamate receptors (iGluRs) mediating excitatory synaptic transmission. Cell surface expressed KARs modulate the excitability of neuronal networks. The transfer of iGluRs from the endoplasmic reticulum (ER) to the cell surface requires occupation of the agonist binding sites. Here we used molecular modelling to produce a range of ligand binding domain (LBD) point mutants of GluK1–3 KAR subunits with and without altered agonist efficacy to further investigate the role of glutamate binding in surface trafficking and activation of homomeric and heteromeric KARs using endoglycosidase digestion, cell surface biotinylation and imaging of changes in intracellular Ca²⁺ concentration [Ca²⁺]_i. Mutations of conserved amino acid residues in the LBD that disrupt agonist binding to GluK1–3 (GluK1-T675V, GluK2-A487L, GluK2-T659V and GluK3-T661V) reduced both the total expression levels and cell surface delivery of all of these mutant subunits compared to the corresponding wild type in transiently transfected human embryonic kidney 293 (HEK293) cells. In contrast, the exchange of non-conserved residues in the LBD that convert antagonist selectivity of GluK1–3 (GluK1-T503A, GluK2-A487T, GluK3-T489A, GluK1-N705S/S706N, GluK2-S689N/N690S, GluK3-N691S) did not alter the biosynthesis and trafficking of subunit proteins. Co-assembly of mutant GluK2 with an impaired LBD and wild type GluK5 subunits enables the cell surface expression of both subunits. However, [Ca²⁺]_i imaging indicates that the occupancy of both GluK2 and GluK5 LBDs is required for the full activation of GluK2/GluK5 heteromeric KAR channels.

     

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

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 GABA_B 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.     

The ligand‐binding domain of a chemoreceptor from Comamonas testosteroni has a previously unknown homotrimeric structure

Transmembrane chemoreceptors are widely present in Bacteria and Archaea. They play a critical role in sensing various signals outside and transmitting to the cell interior. Here, we report the structure of the periplasmic ligand‐binding domain (LBD) of the transmembrane chemoreceptor MCP2201, which governs chemotaxis to citrate and other organic compounds in Comamonas testosteroni. The apo‐form LBD crystal revealed a typical four‐helix bundle homodimer, similar to previously well‐studied chemoreceptors such as Tar and Tsr of Escherichia coli. However, the citrate‐bound LBD revealed a four‐helix bundle homotrimer that had not been observed in bacterial chemoreceptor LBDs. This homotrimer was further confirmed with size‐exclusion chromatography, analytical ultracentrifugation and cross‐linking experiments. The physiological importance of the homotrimer for chemotaxis was demonstrated with site‐directed mutations of key amino acid residues in C. testosteroni mutants.     

Role of Cross-Cleft Contacts in NMDA Receptor Gating

In response to brief glutamate exposure, NMDA receptors produce excitatory currents that have sub-maximal amplitudes and characteristically slow kinetics. The activation sequence starts when glutamate binds to residues located on the upper lobe of extracellularly located ligand-binding domains (LBDs) and then contacts lower lobe residues to bridge the cleft between the two hinged lobes. This event stabilizes a narrow-cleft LBD conformation and may facilitate subsequent inter-lobe contacts that further stabilize the closed cleft. Agonist efficacy has been traced to the degree of agonist-induced cleft-closure and may also depend on the stability of the closed-cleft conformation. To investigate how cross-cleft contacts contribute to the amplitude and kinetics of NMDA receptor response, we examined the activation reaction of GluN1/GluN2A receptors that had single-residue substitutions at the interface between LBD lobes. We found that side-chain truncations at residues of putative contact between lobes increased glutamate efficacy through independent additive mechanisms in GluN1 and GluN2A subunits. In contrast, removing side-chain charge with isosteric substitutions at the same sites decreased glutamate efficacy. These results support the view that in GluN1/GluN2A receptors’ natural interactions between residues on opposing sides of the ligand-binding cleft encode the stability of the glutamate-bound closed-cleft conformations and limit the degree of cleft closure, thus contributing to the sub-maximal response and emblematically slow NMDA receptor deactivation after brief stimulation.     

Effects of three auxin-inducible LBD members on lateral root formation in Arabidopsis thaliana

In Arabidopsis, two AUXIN RESPONSE FACTORs (ARF7 and ARF19) and several Aux/IAAs regulate auxin-induced lateral root (LR) formation. As direct targets of ARF7 and ARF19, LATERAL ORGAN BOUNDARIES DOMAIN 16 (LBD16), LBD29, and LBD18 have a biological function in the formation of lateral roots (LRs). However, the details of the functions of these three LBDs have remained unclear. Each single T-DNA insert mutant has been shown to have slightly fewer LRs than the wild type. We then created a triple mutant, which exhibited a dramatic defect in the LR formation. Our results show that the lbd mutations can lead to impairment in auxin-induced pericycle cell division and in the expression levels of some D-type cyclins (CYCDs). Simultaneously, PLETHORA (PLT) and PIN-FORMED (PIN), which have been well documented to promote cell mitotic activity and are required for auxin response effects, were down-regulated by these lbd mutations. Our results so far indicate that CYCDs, PLT, and PINs are the main targets of the LBDs. We believe that these three LBDs are involved in cell cycle progression of the pericycle in response to auxin. Overexpression of any of these three LBD genes in the triple mutant was found incapable of completely replacing the other two LBDs. The phenotypes of lbd29 mutants were not completely consistent with lbd16 or lbd18 mutants. This indicates that LBD29 may play a distinctive role compared with LBD16 or LBD18 and LBDs might play partially independent roles during the formation of LRs.     

Modeling of peptides connecting the ligand‐binding and transmembrane domains in the GluR2 glutamate receptor

Ligand‐gated Glutamate receptors (GluR) mediate synaptic signals in the nervous system. Ionotropic GluRs of AMPA type, the subject of this study, are tetrameric assemblies of monomer subunits, each of which is constructed in a modular fashion from functional subdomains. The extracellular ligand‐binding domain (LBD) changes its conformation upon binding of an agonist ligand followed by opening of a transmembrane (TM) ion channel. Peptides connecting the LBD and TM domains facilitate gating of the channel, and their structure and composition are important for the receptor functioning. In this study, we used replica exchange molecular dynamics (REMD) simulations to model S1M1 and S2M3 connecting peptides of the GluR2 receptor in two implicit solvents, water and interfacial water/lipid medium characterized by lower polarity. Propensity of these peptides to form helical structures was analyzed using helicity measure derived from the free energy of the simulated ensembles of structures. The S1M1 and S2M3 connecting peptides were not helical in our simulations in both dielectric environments in the absence of the rest of the protein. The structures of the LBD fragment with known high‐resolution α‐helical structure and of the TM3 helix were successfully predicted in the simulations, which in part validate our results. The S2M3 peptide, which is important in gating, formed a well‐defined coil structure and salt‐bridges with the S2 domain. The S1M1 peptide formed a loop structure via formation of internal salt‐bridges. Potential implications of these structures on function of the receptor are discussed. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Principal role of NR3 subunits in NR1/NR3 excitatory glycine receptor function

Calcium-permeable N-methyl-d-aspartate (NMDA) receptors are tetrameric cation channels composed of glycine-binding NR1 and glutamate-binding NR2 subunits, which require binding of both glutamate and glycine for efficient channel gating. In contrast, receptors assembled from NR1 and NR3 subunits function as calcium-impermeable excitatory glycine receptors that respond to agonist application only with low efficacy. Here, we show that antagonists of and substitutions within the glycine-binding site of NR1 potentiate NR1/NR3 receptor function up to 25-fold, but inhibition or mutation of the NR3 glycine binding site reduces or abolishes receptor activation. Thus, glycine bound to the NR1 subunit causes auto-inhibition of NR1/NR3 receptors whereas glycine binding to the NR3 subunits is required for opening of the ion channel. Our results establish differential roles of the high-affinity NR3 and low-affinity NR1 glycine-binding sites in excitatory glycine receptor function.