Expression, purification and characterization of human proton-coupled oligopeptide transporter 1 hPEPT1.

The human peptide transporter hPEPT1 (SLC15A1) is responsible for uptake of dietary di- and tripeptides and a number of drugs from the small intestine by utilizing the proton electrochemical gradient, and hence an important target for peptide-like drug design and drug delivery. hPEPT1 belongs to the ubiquitous major facilitator superfamily that all contain a 12TM core structure, with global conformational changes occurring during the transport cycle. Several bacterial homologues of these transporters have been characterized, providing valuable insight into the transport mechanism of this family. Here we report the overexpression and purification of recombinant hPEPT1 in a detergent-solubilized state. Thermostability profiling of hPEPT1 at different pH values revealed that hPEPT1 is more stable at pH 6 as compared to pH 7 and 8. Micro-scale thermophoresis (MST) confirmed that the purified hPEPT1 was able to bind di- and tripeptides respectively. To assess the in-solution oligomeric state of hPEPT1, negative stain electron microscopy was performed, demonstrating a predominantly monomeric state.

The prototypical proton-coupled oligopeptide transporter YdgR from Escherichia coli facilitates chloramphenicol uptake into bacterial cells.

Chloramphenicol (Cam) is a broad-spectrum antibiotic used to combat bacterial infections in humans and animals. Cam export from bacterial cells is one of the mechanisms by which pathogens resist Cam's antibacterial effects, and several different proteins are known to facilitate this process. However, to date no report exists on any specific transport protein that facilitates Cam uptake. The proton-coupled oligopeptide transporter (POT) YdgR from Escherichia coli is a prototypical member of the POT family, functioning in proton-coupled uptake of di- and tripeptides. By following bacterial growth and conducting LC-MS-based assays we show here that YdgR facilitates Cam uptake. Some YdgR variants displaying reduced peptide uptake also exhibited reduced Cam uptake, indicating that peptides and Cam bind YdgR at similar regions. Homology modeling of YdgR, Cam docking, and mutational studies suggested a binding mode that resembles that of Cam binding to the multidrug resistance transporter MdfA. To our knowledge, this is the first report of Cam uptake into bacterial cells mediated by a specific transporter protein. Our findings suggest a specific bacterial transporter for drug uptake that might be targeted to promote greater antibiotic influx to increase cytoplasmic antibiotic concentration for enhanced cytotoxicity.

Structural Basis of Histone Demethylase KDM6B Histone 3 Lysine 27 Specificity.

KDM subfamily 6 enzymes KDM6A and KDM6B specifically catalyze demethylation of di- and trimethylated lysine on histone 3 lysine 27 (H3K27me3/2) and play an important role in repression of developmental genes. Despite identical amino acid sequence in the immediate surroundings of H3K9me3/2 (ARKS), the enzymes do not catalyze demethylation of this general marker of repression. To address this question for KDM6B, we used computational methods to identify H3(17-33)-derived peptides with improved binding affinity that would allow co-crystallization with the catalytic core of human KDM6B (ccKDM6B). A total of five peptides were identified, and their IC50 values were determined in a matrix-assisted laser desorption ionization time-of-flight-based assay. Despite none of the peptides showing affinity significantly higher than that of the H3(17-33) peptide, it was possible to co-crystallize ccKDM6B with a H3(17-33)A21M peptide. This structure reveals the interactions between the KDM6B zinc binding domain and the H3(17-23) region. Although KDM6A and KDM6B differ in primary sequence, particularly in the H3L20 binding pocket of the zinc binding domains, where two histidines in KDM6A have been replaced by a glutamate and a tyrosine, they bind H3(17-23) in a very similar fashion. This structure shows that KDM6B, in analogy with KDM6A, also uses the zinc binding domain to achieve H3K27me3/me2 specificity. The histidine to glutamine substitution at amino acid position 1564 in the KDM6B zinc binding domain can further explain why KDM6B binds substrates with an affinity higher than that of KDM6A.

Human proton coupled folic acid transporter is a monodisperse oligomer in the lauryl maltose neopentyl glycol solubilized state.

The human proton coupled folic acid transporter PCFT is the major import route for dietary folates. Mutations in the gene encoding PCFT cause hereditary folic acid malabsorption, which manifests itself by compromised folate absorption from the intestine and also in impaired folate transport into the central nervous system. Since its recent discovery, PCFT has been the subject of numerous biochemical studies aiming at understanding its structure and mechanism. One major focus has been its oligomeric state, with some reports supporting oligomers and others a monomer. Here, we report the overexpression and purification of recombinant PCFT. Following detergent screening, n-Dodecyl ß-D-maltoside (DDM) and lauryl maltose neopentyl glycol (LMNG) were chosen for further work as they exhibited the most optimal solubilization. We found that purified detergent solubilized PCFT was able to bind folic acid, thus indicating a functionally active protein. Size exclusion chromatography showed that PCFT in DDM was polydisperse; the LMNG preparation was clearly monodisperse but with shorter retention time than the major DDM peak. To assess the oligomeric state negative stain electron microscopy was performed which showed a particle with the size of a PCFT dimer.

Peptides Derived from Histone 3 and Modified at Position 18 Inhibit Histone Demethylase KDM6 Enzymes.

The KDM6 subfamily of histone lysine demethylases has recently been implicated as a putative target in the treatment of a number of diseases; this makes the availability of potent and selective inhibitors important. Due to high sequence similarity of the catalytic domain of Jumonji C histone demethylases, the development of small-molecule, family-specific inhibitors has, however, proven challenging. One approach to achieve the selective inhibition of these enzymes is the use of peptides derived from the substrate, the histone 3 C terminus. Here we used computational methods to optimize such inhibitors of the KDM6 family. Through natural amino acid substitution, it is shown that a K18I variant of a histone H3 derived peptide significantly increases affinity towards the KDM6 enzymes. The crystal structure of KDM6B in complex with a histone 3 derived K18I peptide reveals a tighter fit of the isoleucine side chain, compared with that of the arginine. As a consequence, the peptide R17 residue also has increased hydrophilic interactions. These interactions of the optimized peptide are likely to be responsible for the increased affinity to the KDM6 enzymes.

Structure-Based Design of a New Scaffold for Cell-Penetrating Peptidic Inhibitors of the Histone Demethylase PHF8.

The histone demethylase PHF8 catalyzes demethylation of mono- and di-methylated Lys9 on histone H3 (H3K9me1/2), and is a transcriptional activator involved in the development and cancer. Affinity and specificity of PHF8 towards H3K9me2 is affected by interaction with both the catalytic domain and a PHD reader domain. The latter specifically recognizes tri-methylated Ly4 on histone H3. A fragment of the histone H3 tail with tri-methylated Lys4 was used as a template for the structure-based design of a cyclic, cell-penetrating peptide that exhibits micromolar binding affinity to PHF8 in biochemical assays. The inhibitor has significantly lower affinity towards KDM2 enzymes (the phylogenetically closest subfamily), and to KDM3 and KDM6 subfamilies. Selectivity is only marginal towards an enzyme from the KDM4 family, which shares histone tail specificity with PHF8. It is a substrate of KDM5B, thus implying that the free N terminus is not part of the KDM5 enzyme substrate recognition machinery. The cyclic peptide's ability to penetrate cells is achieved by incorporation of a sequence derived from HIV Tat. The derived cyclic peptide can be used as a starting compound in the search for potent and selective PHF8 inhibitors.

Expression, purification and characterization of the human MTA2-RBBP7 complex.

The repressive Nucleosome Remodeling and histone Deacetylation (NuRD) complex remodels the chromatin structure by coupling ATP-dependent remodeling activity with histone deacetylase function and plays important roles in regulating gene transcription, DNA damage repair and chromatin assembly. The complex is composed of six subunits: Metastasis Associated proteins MTA1/2/3 initially recruit histone chaperones RBBP4/7 followed by the histone deacetylases HDAC1/2 forming a core complex. Further association of the CpG-binding protein MBD2/3, p66α/ß and the ATP-dependent helicase CDH3/4 constitutes the NuRD complex. Recent structural studies on truncated human proteins or orthologous have revealed that the stoichiometry of the MTA1-RBBP4 complex is 2:4. This study reports expression and purification of the intact human MTA2-RBBP7 complex using HEK293F cells as expression system. In analogy with findings on the Drosophila NuRD complex, we find that also the human MTA-RBBP can be isolated in vitro. Taken together with previous findings this suggests, that MTA-RBBP is a stable complex, with a central role in the initial assembly of the human NuRD complex. Refined 3D volumes of the complex generated from negative stain electron microscopy (EM) data reveals an elongated architecture that is capable of hinge like motion around the center of the particle.

Pharmacology and Structural Analysis of Ligand Binding to the Orthosteric Site of Glutamate-Like GluD2 Receptors.

The GluD2 receptor is a fundamental component of postsynaptic sites in Purkinje neurons, and is required for normal cerebellar function. GluD2 and the closely related GluD1 are classified as members of the ionotropic glutamate receptor (iGluR) superfamily on the basis of sequence similarity, but do not bind l-glutamate. The amino acid neurotransmitter D-Ser is a GluD2 receptor ligand, and endogenous D-Ser signaling through GluD2 has recently been shown to regulate endocytosis of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type iGluRs during synaptic plasticity in the cerebellum, such as long-term depression. Here, we investigate the pharmacology of the orthosteric binding site in GluD2 by examining the activity of analogs of D-Ser and GluN1 glycine site competitive antagonists at GluD2 receptors containing the lurcher mutation (GluD2(LC)), which promotes spontaneous channel activation. We identify several compounds that modulate GluD2(LC), including a halogenated alanine analog as well as the kynurenic acid analog 7-chloro-4-oxo-1H-quinoline-2-carboxylic acid (7-chlorokynurenic acid; 7-CKA). By correlating thermodynamic and structural data for 7-CKA binding to the isolated GluD2 ligand binding domain (GluD2-LBD), we find that binding 7-CKA to GluD2-LBD differs from D-Ser by inducing an intermediate cleft closure of the clamshell-shaped LBD. The GluD2 ligands identified here can potentially serve as a starting point for development of GluD2-selective ligands useful as tools in studies of the signaling role of the GluD2 receptor in the brain.

Acetylcholine-Binding Protein Engineered to Mimic the α4-α4 Binding Pocket in α4ß2 Nicotinic Acetylcholine Receptors Reveals Interface Specific Interactions Important for Binding and Activity.

Neuronal α4ß2 nicotinic acetylcholine receptors are attractive drug targets for psychiatric and neurodegenerative disorders and smoking cessation aids. Recently, a third agonist binding site between two α4 subunits in the (α4)(3)(ß2)(2) receptor subpopulation was discovered. In particular, three residues, H142, Q150, and T152, were demonstrated to be involved in the distinct pharmacology of the α4-α4 versus α4-ß2 binding sites. To obtain insight into the three-dimensional structure of the α4-α4 binding site, a surrogate protein reproducing α4-α4 binding characteristics was constructed by introduction of three point mutations, R104H, L112Q, and M114T, into the binding pocket of Lymnaea stagnalis acetylcholine-binding protein (Ls-AChBP). Cocrystallization with two agonists possessing distinct pharmacologic profiles, NS3920 [1-(6-bromopyridin-3-yl)-1,4-diazepane] and NS3573 [1-(5-ethoxypyridin-3-yl)-1,4-diazepane], highlights the roles of the three residues in determining binding affinities and functional properties of ligands at the α4-α4 interface. Confirmed by mutational studies, our structures suggest a unique ligand-specific role of residue H142 on the α4 subunit. In the cocrystal structure of the mutated Ls-AChBP with the high-efficacy ligand NS3920, the corresponding histidine forms an intersubunit bridge that reinforces the ligand-mediated interactions between subunits. The structures further reveal that the binding site residues gain different and ligand-dependent interactions that could not be predicted based on wild-type Ls-AChBP structures in complex with the same agonists. The results show that an unprecedented correlation between binding in engineered AChBPs and functional receptors can be obtained and provide new opportunities for structure-based design of drugs targeting specific nicotinic acetylcholine receptor interfaces.

Structural and functional studies of the modulator NS9283 reveal agonist-like mechanism of action at α4ß2 nicotinic acetylcholine receptors.

Modulation of Cys loop receptor ion channels is a proven drug discovery strategy, but many underlying mechanisms of the mode of action are poorly understood. We report the x-ray structure of the acetylcholine-binding protein from Lymnaea stagnalis with NS9283, a stoichiometry selective positive modulator that targets the α4-α4 interface of α4ß2 nicotinic acetylcholine receptors (nAChRs). Together with homology modeling, mutational data, quantum mechanical calculations, and pharmacological studies on α4ß2 nAChRs, the structure reveals a modulator binding mode that overlaps the α4-α4 interface agonist (acetylcholine)-binding site. Analysis of contacts to residues known to govern agonist binding and function suggests that modulation occurs by an agonist-like mechanism. Selectivity for α4-α4 over α4-ß2 interfaces is determined mainly by steric restrictions from Val-136 on the ß2-subunit and favorable interactions between NS9283 and His-142 at the complementary side of α4. In the concentration ranges where modulation is observed, its selectivity prevents NS9283 from directly activating nAChRs because activation requires coordinated action from more than one interface. However, we demonstrate that in a mutant receptor with one natural and two engineered α4-α4 interfaces, NS9283 is an agonist. Modulation via extracellular binding sites is well known for benzodiazepines acting at γ-aminobutyric acid type A receptors. Like NS9283, benzodiazepines increase the apparent agonist potency with a minimal effect on efficacy. The shared modulatory profile along with a binding site located in an extracellular subunit interface suggest that modulation via an agonist-like mechanism may be a common mechanism of action that potentially could apply to Cys loop receptors beyond the α4ß2 nAChRs.

A high-affinity, dimeric inhibitor of PSD-95 bivalently interacts with PDZ1-2 and protects against ischemic brain damage.

Inhibition of the ternary protein complex of the synaptic scaffolding protein postsynaptic density protein-95 (PSD-95), neuronal nitric oxide synthase (nNOS), and the N-methyl-D-aspartate (NMDA) receptor is a potential strategy for treating ischemic brain damage, but high-affinity inhibitors are lacking. Here we report the design and synthesis of a novel dimeric inhibitor, Tat-NPEG4(IETDV)(2) (Tat-N-dimer), which binds the tandem PDZ1-2 domain of PSD-95 with an unprecedented high affinity of 4.6 nM, and displays extensive protease-resistance as evaluated in vitro by stability-measurements in human blood plasma. X-ray crystallography, NMR, and small-angle X-ray scattering (SAXS) deduced a true bivalent interaction between dimeric inhibitor and PDZ1-2, and also provided a dynamic model of the conformational changes of PDZ1-2 induced by the dimeric inhibitor. A single intravenous injection of Tat-N-dimer (3 nmol/g) to mice subjected to focal cerebral ischemia reduces infarct volume with 40% and restores motor functions. Thus, Tat-N-dimer is a highly efficacious neuroprotective agent with therapeutic potential in stroke.

Two distinct allosteric binding sites at α4ß2 nicotinic acetylcholine receptors revealed by NS206 and NS9283 give unique insights to binding activity-associated linkage at Cys-loop receptors.

Positive allosteric modulators (PAMs) of α4ß2 nicotinic acetylcholine receptors have the potential to improve cognitive function and alleviate pain. However, only a few selective PAMs of α4ß2 receptors have been described limiting both pharmacological understanding and drug-discovery efforts. Here, we describe a novel selective PAM of α4ß2 receptors, NS206, and compare with a previously reported PAM, NS9283. Using two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes, NS206 was observed to positively modulate acetylcholine (ACh)-evoked currents at both known α4ß2 stoichiometries (2α:3ß and 3α:2ß). In the presence of NS206, peak current amplitudes surpassed those of maximal efficacious ACh stimulations (Emax(ACh)) with no or limited effects at potencies and current waveforms (as inspected visually). This pharmacological action contrasted with that of NS9283, which only modulated the 3α:2ß receptor and acted by left shifting the ACh concentration-response relationship. Interestingly, the two modulators can act simultaneously in an additive manner at 3α:2ß receptors, which results in current levels exceeding Emax(ACh) and a left-shifted ACh concentration-response relationship. Through use of chimeric and point-mutated receptors, the binding site of NS206 was linked to the α4-subunit transmembrane domain, whereas binding of NS9283 was shown to be associated with the αα-interface in 3α:2ß receptors. Collectively, these data demonstrate the existence of two distinct modulatory sites in α4ß2 receptors with unique pharmacological attributes that can act additively. Several allosteric sites have been identified within the family of Cys-loop receptors and with the present data, a detailed picture of allosteric modulatory mechanisms of these important receptors is emerging.

Molecular determinants of subtype-selective efficacies of cytisine and the novel compound NS3861 at heteromeric nicotinic acetylcholine receptors.

Deciphering which specific agonist-receptor interactions affect efficacy levels is of high importance, because this will ultimately aid in designing selective drugs. The novel compound NS3861 and cytisine are agonists of nicotinic acetylcholine receptors (nAChRs) and both bind with high affinity to heteromeric α3ß4 and α4ß2 nAChRs. However, initial data revealed that the activation patterns of the two compounds show very distinct maximal efficacy readouts at various heteromeric nAChRs. To investigate the molecular determinants behind these observations, we performed in-depth patch clamp electrophysiological measurements of efficacy levels at heteromeric combinations of α3- and α4-, with ß2- and ß4-subunits, and various chimeric constructs thereof. Compared with cytisine, which selectively activates receptors containing ß4- but not ß2-subunits, NS3861 displays the opposite ß-subunit preference and a complete lack of activation at α4-containing receptors. The maximal efficacy of NS3861 appeared solely dependent on the nature of the ligand-binding domain, whereas efficacy of cytisine was additionally affected by the nature of the ß-subunit transmembrane domain. Molecular docking to nAChR subtype homology models suggests agonist specific interactions to two different residues on the complementary subunits as responsible for the ß-subunit preference of both compounds. Furthermore, a principal subunit serine to threonine substitution may explain the lack of NS3861 activation at α4-containing receptors. In conclusion, our results are consistent with a hypothesis where agonist interactions with the principal subunit (α) primarily determine binding affinity, whereas interactions with key amino acids at the complementary subunit (ß) affect agonist efficacy.

Crystal structure and pharmacological characterization of a novel N-methyl-D-aspartate (NMDA) receptor antagonist at the GluN1 glycine binding site.

NMDA receptors are ligand-gated ion channels that mediate excitatory neurotransmission in the brain. They are tetrameric complexes composed of glycine-binding GluN1 and GluN3 subunits together with glutamate-binding GluN2 subunits. Subunit-selective antagonists that discriminate between the glycine sites of GluN1 and GluN3 subunits would be valuable pharmacological tools for studies on the function and physiological roles of NMDA receptor subtypes. In a virtual screening for antagonists that exploit differences in the orthosteric binding site of GluN1 and GluN3 subunits, we identified a novel glycine site antagonist, 1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]quinoxalin-4(5H)-one (TK40). Here, we show by Schild analysis that TK40 is a potent competitive antagonist with Kb values of 21-63 nM at the GluN1 glycine-binding site of the four recombinant GluN1/N2A-D receptors. In addition, TK40 displayed >100-fold selectivity for GluN1/N2 NMDA receptors over GluN3A- and GluN3B-containing NMDA receptors and no appreciable effects at AMPA receptors. Binding experiments on rat brain membranes and the purified GluN1 ligand-binding domain using glycine site GluN1 radioligands further confirmed the competitive interaction and high potency. To delineate the binding mechanism, we have solved the crystal structure of the GluN1 ligand-binding domain in complex with TK40 and show that TK40 binds to the orthosteric binding site of the GluN1 subunit with a binding mode that was also predicted by virtual screening. Furthermore, the structure reveals that the imino acetamido group of TK40 acts as an α-amino acid bioisostere, which could be of importance in bioisosteric replacement strategies for future ligand design.

Structural basis of Δ9-THC analog activity at the Cannabinoid 1 receptor.

Δ9-tetrahydrocannabinol (THC) is the principal psychoactive compound derived from the cannabis plant Cannabis sativa and approved for emetic conditions, appetite stimulation and sleep apnea relief. THC's psychoactive actions are mediated primarily by the cannabinoid receptor CB1. Here, we determine the cryo-EM structure of HU210, a THC analog and widely used tool compound, bound to CB1 and its primary transducer, Gi1. We leverage this structure for docking and 1,000 ns molecular dynamics simulations of THC and 10 structural analogs delineating their spatiotemporal interactions at the molecular level. Furthermore, we pharmacologically profile their recruitment of Gi and ß-arrestins and reversibility of binding from an active complex. By combining detailed CB1 structural information with molecular models and signaling data we uncover the differential spatiotemporal interactions these ligands make to receptors governing potency, efficacy, bias and kinetics. This may help explain the actions of abused substances, advance fundamental receptor activation studies and design better medicines.

Small-molecule positive allosteric modulation of homomeric kainate receptors GluK1-3: development of screening assays and insight into GluK3 structure.

The kainate receptors GluK1-3 (glutamate receptor ionotropic, kainate receptors 1-3) belong to the family of ionotropic glutamate receptors and are essential for fast excitatory neurotransmission in the brain, and are associated with neurological and psychiatric diseases. How these receptors can be modulated by small-molecule agents is not well understood, especially for GluK3. We show that the positive allosteric modulator BPAM344 can be used to establish robust calcium-sensitive fluorescence-based assays to test agonists, antagonists, and positive allosteric modulators of GluK1-3. The half-maximal effective concentration (EC50) of BPAM344 for potentiating the response of 100 µm kainate was determined to be 26.3 µm for GluK1, 75.4 µm for GluK2, and 639 µm for GluK3. Domoate was found to be a potent agonist for GluK1 and GluK2, with an EC50 of 0.77 and 1.33 µm, respectively, upon co-application of 150 µm BPAM344. At GluK3, domoate acts as a very weak agonist or antagonist with a half-maximal inhibitory concentration (IC50) of 14.5 µm, in presence of 500 µm BPAM344 and 100 µm kainate for competition binding. Using H523A-mutated GluK3, we determined the first dimeric structure of the ligand-binding domain by X-ray crystallography, allowing location of BPAM344, as well as zinc-, sodium-, and chloride-ion binding sites at the dimer interface. Molecular dynamics simulations support the stability of the ion sites as well as the involvement of Asp761, Asp790, and Glu797 in the binding of zinc ions. Using electron microscopy, we show that, in presence of glutamate and BPAM344, full-length GluK3 adopts a dimer-of-dimers arrangement.

Intersubunit bridge formation governs agonist efficacy at nicotinic acetylcholine α4ß2 receptors: unique role of halogen bonding revealed.

The α4ß2 subtype of the nicotinic acetylcholine receptor has been pursued as a drug target for treatment of psychiatric and neurodegenerative disorders and smoking cessation aids for decades. Still, a thorough understanding of structure-function relationships of α4ß2 agonists is lacking. Using binding experiments, electrophysiology and x-ray crystallography we have investigated a consecutive series of five prototypical pyridine-containing agonists derived from 1-(pyridin-3-yl)-1,4-diazepane. A correlation between binding affinities at α4ß2 and the acetylcholine-binding protein from Lymnaea stagnalis (Ls-AChBP) confirms Ls-AChBP as structural surrogate for α4ß2 receptors. Crystal structures of five agonists with efficacies at α4ß2 from 21-76% were determined in complex with Ls-AChBP. No variation in closure of loop C is observed despite large efficacy variations. Instead, the efficacy of a compound appears tightly coupled to its ability to form a strong intersubunit bridge linking the primary and complementary binding interfaces. For the tested agonists, a specific halogen bond was observed to play a large role in establishing such strong intersubunit anchoring.

Interaction partners of PSD-93 studied by X-ray crystallography and fluorescence polarization spectroscopy.

PSD-93 (chapsyn-110, DLG2) is a member of the family of membrane-associated guanylate kinase (MAGUK) proteins. The MAGUK proteins are involved in receptor localization and signalling pathways. The best characterized MAGUK protein, PSD-95, is known to be involved in NMDA receptor signalling via its PDZ domains. The PDZ domains of PSD-95 and PSD-93 are structurally very similar, but relatively little is known about the function of PSD-93. PSD-93 has been suggested to interact with GluD2 from the family of ionotropic glutamate receptors. Here, the interactions of four residues (GTSI) representing the extreme C-terminus of GluD2 with PSD-93 PDZ1 have been investigated in the crystalline phase. Two different binding modes of these residues were observed, suggesting that the peptide is not tightly bound to PSD-93 PDZ1. In accordance, the two N-terminal PSD-93 PDZ domains show no appreciable binding affinity for a GluD2-derived C-terminal octapeptide, whereas micromolar affinity was observed for a GluN2B-derived C-terminal octapeptide. This indicates that if present, the interactions between GluD2 and PSD-93 involve more than the extreme terminus of the receptor. In contrast, the tumour-suppressor protein SCRIB PDZ3 shows low micromolar affinity towards the GluD2-derived octapeptide, which is in agreement with previous findings using high-throughput assays.

Structural analysis of the positive AMPA receptor modulators CX516 and Me-CX516 in complex with the GluA2 ligand-binding domain.

Positive allosteric modulators of the ionotropic glutamate receptor A2 (GluA2) can serve as lead compounds for the development of cognitive enhancers. Several benzamide-type (S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptor modulators such as aniracetam, CX516 and CX614 have been shown to inhibit the deactivation of AMPA receptors with a less pronounced effect on desensitization. Despite CX516 being an extensively investigated AMPA receptor modulator and one of the few clinically evaluated compounds, the binding mode of CX516 to AMPA receptors has not been reported. Here, the structures of a GluA2 ligand-binding domain mutant in complex with CX516 and the 3-methylpiperidine analogue of CX516 (Me-CX516) are reported. The structures show that the binding modes of CX516 and Me-CX516 are similar to those of aniracetam and CX614 and that there is limited space for substitution at the piperidine ring of CX516. The results therefore support that CX516, like aniracetam and CX614, modulates deactivation of AMPA receptors.

The structure of amylosucrase from Deinococcus radiodurans has an unusual open active-site topology.

Amylosucrases (ASes) catalyze the formation of an α-1,4-glucosidic linkage by transferring a glucosyl unit from sucrose onto an acceptor α-1,4-glucan. To date, several ligand-bound crystal structures of wild-type and mutant ASes from Neisseria polysaccharea and Deinococcus geothermalis have been solved. These structures all display a very similar overall conformation with a deep pocket leading to the site for transglucosylation, subsite -1. This has led to speculation on how sucrose enters the active site during glucan elongation. In contrast to previous studies, the AS structure from D. radiodurans presented here has a completely empty -1 subsite. This structure is strikingly different from other AS structures, as an active-site-lining loop comprising residues Leu214-Asn225 is found in a previously unobserved conformation. In addition, a large loop harbouring the conserved active-site residues Asp133 and Tyr136 is disordered. The result of the changed loop conformations is that the active-site topology is radically changed, leaving subsite -1 exposed and partially dismantled. This structure provides novel insights into the dynamics of ASes and comprises the first structural support for an elongation mechanism that involves considerable conformational changes to modulate accessibility to the sucrose-binding site and thereby allows successive cycles of glucosyl-moiety transfer to a growing glucan chain.