Ligand-binding Domain Dimer Interface Research Articles

Positive and negative allosteric modulation of GluK2 kainate receptors by BPAM344 and antiepileptic perampanel.

Kainate receptors (KARs) are a subtype of ionotropic glutamate receptors that control synaptic transmission in the central nervous system and are implicated in neurological, psychiatric, and neurodevelopmental disorders. Understanding the regulation of KAR function by small molecules is essential for exploring these receptors as drug targets. Here, we present cryoelectron microscopy (cryo-EM) structures of KAR GluK2 in complex with the positive allosteric modulator BPAM344, competitive antagonist DNQX, and negative allosteric modulator, antiepileptic drug perampanel. Our structures show that two BPAM344 molecules bind per ligand-binding domain dimer interface. In the absence of an agonist or in the presence of DNQX, BPAM344 stabilizes GluK2 in the closed state. The closed state is also stabilized by perampanel, which binds to the ion channel extracellular collar sites located in two out of four GluK2 subunits. The molecular mechanisms of positive and negative allosteric modulation of KAR provide a guide for developing new therapeutic strategies.

The androgen receptor depends on ligand-binding domain dimerization for transcriptional activation.

Whereas dimerization of the DNA-binding domain of the androgen receptor (AR) plays an evident role in recognizing bipartite response elements, the contribution of the dimerization of the ligand-binding domain (LBD) to the correct functioning of the AR remains unclear. Here, we describe a mouse model with disrupted dimerization of the AR LBD (ARLmon/Y ). The disruptive effect of the mutation is demonstrated by the feminized phenotype, absence of male accessory sex glands, and strongly affected spermatogenesis, despite high circulating levels of testosterone. Testosterone replacement studies in orchidectomized mice demonstrate that androgen-regulated transcriptomes in ARLmon/Y mice are completely lost. The mutated AR still translocates to the nucleus and binds chromatin, but does not bind to specific AR binding sites. In vitro studies reveal that the mutation in the LBD dimer interface also affects other AR functions such as DNA binding, ligand binding, and co-regulator binding. In conclusion, LBD dimerization is crucial for the development of AR-dependent tissues through its role in transcriptional regulation invivo. Our findings identify AR LBD dimerization as a possible target for AR inhibition.

Mechanism of AMPA Receptor Modulation by Gamma-8

Fast excitatory neurotransmission is mediated by ionotropic glutamate receptor (iGluR). AMPA-subtype ionotropic glutamate receptors (AMPARs) are associated with auxiliary subunits (transmembrane AMPA receptor regulatory proteins (TARPs), cornichons, cysteine knot proteins and GSG1); which regulate AMPAR assembly, trafficking, gating, and pharmacology. Of particular interest is the interactions with gamma-8 which results in re-sensitization of the currents where the transmembrane channel in the receptor initially undergoes closure due to desensitization but reopens at longer time and remain open in the continued presence of agonist. Here we have investigated the mechanism underlying this resensitization process using single molecule FRET and single channel functional recordings. Specifically we have determined changes at the AMPA receptor ligand binding domain dimer interface in presence of gamma-8 and compared it to the changes at this site observed in the presence of cyclothiazide, a drug that stabilizes the open channel state of the receptor. These studies show that while the overall states populated are similar in the two cases, the kinetics of transitions between states are different, with activation barrier between transitions being higher in the presence of gamma-8 . The single channel current recordings reflected these slower kinetics in the presence of gamma-8 and also showed very rapid channel openings and closing which were no reflected at the dimer interface suggesting that these may be localized changes at the transmembrane segments.

Enhancing Action of Positive Allosteric Modulators through the Design of Dimeric Compounds.

The present study describes the identification of highly potent dimeric 1,2,4-benzothiadiazine 1,1-dioxide (BTD)-type positive allosteric modulators of the AMPA receptors (AMPApams) obtained by linking two monomeric BTD scaffolds through their respective 6-positions. Using previous X-ray data from monomeric BTDs cocrystallized with the GluA2 ligand-binding domain (LBD), a molecular modeling approach was performed to predict the preferred dimeric combinations. Two 6,6-ethylene-linked dimeric BTD compounds (16 and 22) were prepared and evaluated as AMPApams on HEK293 cells expressing GluA2o( Q) (calcium flux experiment). These compounds were found to be about 10,000 times more potent than their respective monomers, the most active dimeric compound being the bis-4-cyclopropyl-substituted compound 22 [6,6'-(ethane-1,2-diyl)bis(4-cyclopropyl-3,4-dihydro-2 H-1,2,4-benzothiadiazine 1,1-dioxide], with an EC50 value of 1.4 nM. As a proof of concept, the bis-4-methyl-substituted dimeric compound 16 (EC50 = 13 nM) was successfully cocrystallized with the GluA2o-LBD and was found to occupy the two BTD binding sites at the LBD dimer interface.

Structural Basis for Polyamine Binding at the dCACHE Domain of the McpU Chemoreceptor from Pseudomonas putida

Many bacteria can move chemotactically to a variety of compounds and the recognition of chemoeffectors by the chemoreceptor ligand binding domain (LBD) defines the specificity of response. Many chemoreceptors were found to recognize different amino and organic acids, but the McpU chemoreceptor from Pseudomonas putida was identified as the first chemoreceptor that bound specifically polyamines. We report here the three-dimensional structure of McpU-LBD in complex with putrescine at a resolution of 2.4 Å, which fitted well a solution structure generated by small-angle X-ray scattering. Putrescine bound to a negatively charged pocket in the membrane distal module of McpU-LBD. Similarities exist in the binding of putrescine to McpU-LBD and taurine to the LBD of the Mlp37 chemoreceptor of Vibrio cholerae. In both structures, the primary amino group of the respective ligand is recognized by hydrogen bonds established by two aspartate and a tyrosine side chain. This feature may be used to predict the ligands of chemoreceptors with unknown function. Analytical ultracentrifugation revealed that McpU-LBD is monomeric in solution and that ligand binding does not alter this oligomeric state. This sensing mode thus differs from that of the well-characterised four-helix bundle domains where ligands bind to two sites at the LBD dimer interface. Although there appear to be different sensing modes, results are discussed in the context of data, indicating that chemoreceptors employ the same mechanism of transmembrane signaling. This work enhances our understanding of CACHE domains, which are the most abundant sensor domains in bacterial chemoreceptors and sensor kinases.

Synergistic Regulation of Coregulator/Nuclear Receptor Interaction by Ligand and DNA

Nuclear receptor (NR) transcription factors bind various coreceptors, small-molecule ligands, DNA response element sequences, and transcriptional coregulator proteins to affect gene transcription. Small-molecule ligands and DNA are known to influence receptor structure, coregulator protein interaction, and function; however, little is known on the mechanism of synergy between ligand and DNA. Using quantitative biochemical, biophysical, and solution structural methods, including 13C-detected nuclear magnetic resonance and hydrogen/deuterium exchange (HDX) mass spectrometry, we show that ligand and DNA cooperatively recruit the intrinsically disordered steroid receptor coactivator-2 (SRC-2/TIF2/GRIP1/NCoA-2) receptor interaction domain to peroxisome proliferator-activated receptor gamma-retinoid X receptor alpha (PPARγ-RXRα) heterodimer and reveal the binding determinants of the complex. Our data reveal a thermodynamic mechanism by which DNA binding propagates a conformational change in PPARγ-RXRα, stabilizes the receptor ligand binding domain dimer interface, and impacts ligand potency and cooperativity in NR coactivator recruitment.

Architecture of fully occupied GluA2 AMPA receptor–TARP complex elucidated by cryo-EM

Fast excitatory neurotransmission in the mammalian central nervous system is largely carried out by AMPA-sensitive ionotropic glutamate receptors. Localized within the postsynaptic density of glutamatergic spines, AMPA receptors are composed of heterotetrameric receptor assemblies associated with auxiliary subunits, the most common of which are transmembrane AMPA receptor regulatory proteins (TARPs). The association of TARPs with AMPA receptors modulates receptor trafficking and the kinetics of receptor gating and pharmacology. Here we report the cryo-electron microscopy (cryo-EM) structure of the homomeric rat GluA2 AMPA receptor saturated with TARP γ2 subunits, which shows how the TARPs are arranged with four-fold symmetry around the ion channel domain and make extensive interactions with the M1, M2 and M4 transmembrane helices. Poised like partially opened ‘hands’ underneath the two-fold symmetric ligand-binding domain (LBD) 'clamshells', one pair of TARPs is juxtaposed near the LBD dimer interface, whereas the other pair is near the LBD dimer-dimer interface. The extracellular ‘domains’ of TARP are positioned to not only modulate LBD clamshell closure, but also affect conformational rearrangements of the LBD layer associated with receptor activation and desensitization, while the TARP transmembrane domains buttress the ion channel pore.

Mechanism of AMPA Receptor Gating Re-Shaped by Auxiliary Proteins

Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that mediate excitation in the mammalian central nervous system. Amongst iGluRs, the AMPA receptor (AMPAR) subfamily facilitates the initial depolarization of postsynaptic terminals by gating on a millisecond time scale in response to neurotransmitter binding. In recent years, intact structures of the GluA2 AMPAR determined in several conformational states have been used to develop rudimentary structural models of receptor activation and desensitization. However, it is unclear how such models extrapolate to native AMPARs, which are typically associated with “auxiliary” subunits from one or more protein families. Although auxiliary proteins are known to regulate AMPAR plasma membrane expression and function, the sites at which they functionally interact with receptor subunits have yet to identified and/or characterized. Because we previously demonstrated that the apex of the extracellular ligand-binding domain (LBD) dimer interface is critical for iGluR activation, we investigated whether this site similarly regulates AMPAR-auxiliary protein complexes. GluA2 AMPARs were expressed alone or with auxiliary proteins, and patch-clamp electrophysiology was utilized to assess the functional properties of receptors. Our data reveal that an electrostatic network at the apex of the AMPAR LBD is critical for channel activation, since truncation of residues in this network to alanines generated largely nonfunctional receptors. Interestingly, co-expression of these receptors with auxiliary proteins restored channel gating, indicative that gating was, in part, mediated by interactions outside the LBD dimer interface. Through additional mutagenesis we identified a site, distant from the dimer interface, through which the transmembrane AMPA receptor regulatory protein (TARP) class of auxiliary proteins regulates AMPAR gating, but not permeation properties. In conclusion, our results suggest that the activation of native AMPAR complexes is coordinated by interactions within pore-forming subunits, as well as distinct interactions with auxiliary subunits.

Identification of critical functional determinants of kainate receptor modulation by auxiliary protein Neto2.

Kainate receptors (KARs) are ionotropic glutamate receptors (iGluRs) that modulate synaptic transmission and intrinsic neuronal excitability. KARs associate with the auxiliary proteins neuropilin- and tolloid-like 1 and 2 (Neto1 and Neto2), which act as allosteric modulators of receptor function impacting all biophysical properties of these receptors studied to date. M3-S2 linkers play a critical role in KAR gating; we found that individual residues in these linkers bidirectionally influence Neto2 modulation of KAR desensitization in an agonist specific manner. We also identify the D1 dimer interface as a novel site of Neto2 modulation and functionally correlate the actions of Neto2 modulation of desensitization with modulation of cation sensitivity. We identify these domains as determinants of Neto2 modulation. Thus, our work contributes to the understanding of auxiliary subunit modulation of KAR function and could aid the development of KAR-specific modulators to alter receptor function. Kainate receptors (KARs) are important modulators of synaptic transmission and intrinsic neuronal excitability in the CNS. Their activity is shaped by the auxiliary proteins Neto1 and Neto2, which impact KAR gating in a receptor subunit- and Neto isoform-specific manner. The structural basis for Neto modulation of KAR gating is unknown. Here we identify the M3-S2 gating linker as a critical determinant contributing to Neto2 modulation of KARs. M3-S2 linkers control both the valence and magnitude of Neto2 modulation of homomeric GluK2 receptors. Furthermore, a single mutation in this domain abolishes Neto2 modulation of heteromeric receptor desensitization. Additionally, we found that cation sensitivity of KAR gating is altered by Neto2 association, suggesting that stability of the D1 dimer interface in the ligand-binding domain (LBD) is an important determinant of Neto2 actions. Moreover, modulation of cation sensitivity was eliminated by mutations in the M3-S2 linkers, thereby correlating the action of Neto2 at these structurally discrete sites on receptor subunits. These results demonstrate that the KAR M3-S2 linkers and LBD dimer interface are critical determinants for Neto2 modulation of receptor function and identify these domains as potential sites of action for the targeted development of KAR-specific modulators that alter the function of auxiliary proteins in native receptors.

Regulatory Ions Bound at the iGluR Ligand Binding Domain Dimer Interface - A Shared Property of GluK2 and AvGluR1?

Ligand-gated ion channels activated by glutamate binding, ionotropic glutamate receptor (iGluRs), play crucial roles in our central nervous system (CNS), e.g. in learning and memory. Furthermore, iGluRs are implicated in many CNS disorders. Binding of glutamate to the iGluR ligand-binding domain (LBD) triggers opening of the transmembrane cation channel. In the overall tetrameric structure, the LBDs are organized in a dimer of dimers. As opposed to other vertebrate iGluRs, kainate-selective iGluRs (KARs) require binding of extracellular sodium and chloride ions to the LBD dimer interface in addition to agonist binding for activation. We have recently shown that the regulatory cations for the GluK2 KAR determine the onset of desensitization, with desensitization not occurring until the cation site is vacated (Nat. Struct. Mol. Biol. 2013, 20, 1054-1061). iGluRs are also found in other kingdoms of life, structurally exemplified by AvGluR1 from a primitive eukaryote (Structure 2013, 21, 1-12). Surprisingly, this LBD dimer structure shows four chloride ions bound at the dimer interface. With atomistic molecular dynamics simulations similar to our work on KARs, we have studied the binding of these chloride ions to elucidate whether they appear to have a regulatory effect on AvGluR1, and whether such a regulatory mechanism could be a shared property between KARs and AvGluR1. Furthermore, we have studied the structural changes believed to be linked to desensitization in the presence and absence of interface-bound ions for both structures. The LBD dimer interface is thought to open up around the ion binding sites in conjunction with desensitization. Interestingly, this interface is packed closer in the AvGluR1 structure than observed for LBD dimer structures of vertebrate iGluRs. Our results illustrate how this feature influences the dynamical changes of desensitization.

Inhibitory Effects of 2-Naphthoic Acid on the Gating of NMDA Receptors

N-methyl-D-aspartate (NMDA) receptors mediate excitatory synaptic transmission in central nervous system and play important roles in development and synaptic plasticity, but have also been found to mediate neurotoxicity. Recently, 2-naphthoic acid (NPA) and its derivatives have been identified as allosteric, noncompetitive NMDA receptors inhibitors. The inhibitory selectivity of NPA derivatives among NMDA receptor subtypes was mapped to the ligand-binding domain (LBD), and the binding site of NPA is proposed to be located in the LBD dimer interface. However, the mechanism by which NPA exerts inhibitory effect is still unclear. We examined how NPA affects the gating reaction of NMDA receptors. Whole-cell patch clamp on HEK 293 cells expressing recombinant NR1-1a/NR2A showed that NPA has 90% maximum inhibition with IC50 of 2.1 mM. Furthermore, by recording single-channel currents from NR1-1a/NR2A receptors with 4 mM NPA, we found a 62% decrease in open probability (Po), due to a 2.5-fold decrease in mean open time (MOT) and a 2-fold increase in mean closed time (MCT). Kinetic modeling suggests NPA increases energy barrier of gating and destabilizes the open state, thus making NRs accumulate in the closed states along the activation pathway. These results provide insight into the inhibitory mechanism of NPA, and help anticipate its effects on both physiological and pathological conditions.