Ligand Binding Properties Research Articles

Uncovering the Domain-Specific Interactome of the TAF1 Tandem Reader Using Site-Specific Azide-Acetyllysine Photochemistry.

Combinatorial readout of histone post-translational modifications by tandem reader modules mediates crosstalk among different histone modifications. To identify the domain-specific interactome of the tandem reader, we engineered the dual bromodomain of TATA-binding protein-associated factor-1 (TAF1) to carry a photoactivatable unnatural amino acid, 4-azido-l-phenylalanine (AzF), via amber suppressor mutagenesis. Using computational approaches, we modeled the targeted residues of TAF1 with AzF to predict the cross-linking distance between the reactive arylazide and its interacting partner. We developed three photoactivatable TAF1 tandem-bromodomain analogues, viz., Y1403AzF in bromodomain 1 (BD1), W1526AzF in bromodomain 2 (BD2), and Y1403AzF/W1526AzF in both BD1 and BD2. Circular dichroism and a thermal shift assay were used to confirm the structural integrity of the engineered readers. Using the TAF1 tandem-bromodomain analogues, we characterized their histone ligand binding properties by isothermal titration calorimetry and photo-cross-linking experiments. We found that the dual bromodomain of TAF1 independently binds and cross-links to different acetylated histone ligands. We further used the engineered BD1 and BD2 analogues of the TAF1 tandem readers to identify their domain-specific interacting partners at the cellular level. Both BD1 and BD2 independently cross-link to a unique interactome, and importantly, the dual cross-linker carrying TAF1 analogue could capture both BD1- and BD2-specific interactomes. Our work concludes that BD1 and BD2 of the TAF1 tandem reader independently recognize their interacting partners to regulate downstream cellular functions.

Coordination chemogenetics for activation of GPCR-type glutamate receptors in brain tissue

Direct activation of cell-surface receptors is highly desirable for elucidating their physiological roles. A potential approach for cell-type-specific activation of a receptor subtype is chemogenetics, in which both point mutagenesis of the receptors and designed ligands are used. However, ligand-binding properties are affected in most cases. Here, we developed a chemogenetic method for direct activation of metabotropic glutamate receptor 1 (mGlu1), which plays essential roles in cerebellar functions in the brain. Our screening identified a mGlu1 mutant, mGlu1(N264H), that was activated directly by palladium complexes. A palladium complex showing low cytotoxicity successfully activated mGlu1 in mGlu1(N264H) knock-in mice, revealing that activation of endogenous mGlu1 is sufficient to evoke the critical cellular mechanism of synaptic plasticity, a basis of motor learning in the cerebellum. Moreover, cell-type-specific activation of mGlu1 was demonstrated successfully using adeno-associated viruses in mice, which shows the potential utility of this chemogenetics for clarifying the physiological roles of mGlu1 in a cell-type-specific manner.

How insulin-like growth factor I binds to a hybrid insulin receptor type 1 insulin-like growth factor receptor

SummaryMonomers of the insulin receptor and type 1 insulin-like growth factor receptor (IGF-1R) can combine stochastically to form heterodimeric hybrid receptors. These hybrid receptors display ligand binding and signaling properties that differ from those of the homodimeric receptors. Here, we describe the cryoelectron microscopy structure of such a hybrid receptor in complex with insulin-like growth factor I (IGF-I). The structure (ca. 3.7 Å resolution) displays a single IGF-I ligand, bound in a similar fashion to that seen for IGFs in complex with IGF-1R. The IGF-I ligand engages the first leucine-rich-repeat domain and cysteine-rich region of the IGF-1R monomer (rather than those of the insulin receptor monomer), consistent with the determinants for IGF binding residing in the IGF-1R cysteine-rich region. The structure broadens our understanding of this receptor family and assists in delineating the key structural motifs involved in binding their respective ligands.

Live-cell microscopy or fluorescence anisotropy with budded baculoviruses-which way to go with measuring ligand binding to M4 muscarinic receptors?

M4 muscarinic acetylcholine receptor is a G protein-coupled receptor (GPCR) that has been associated with alcohol and cocaine abuse, Alzheimer's disease, and schizophrenia which makes it an interesting drug target. For many GPCRs, the high-affinity fluorescence ligands have expanded the options for high-throughput screening of drug candidates and serve as useful tools in fundamental receptor research. Here, we explored two TAMRA-labelled fluorescence ligands, UR-MK342 and UR-CG072, for development of assays for studying ligand-binding properties to M4 receptor. Using budded baculovirus particles as M4 receptor preparation and fluorescence anisotropy method, we measured the affinities and binding kinetics of both fluorescence ligands. Using the fluorescence ligands as reporter probes, the binding affinities of unlabelled ligands could be determined. Based on these results, we took a step towards a more natural system and developed a method using live CHO-K1-hM4R cells and automated fluorescence microscopy suitable for the routine determination of unlabelled ligand affinities. For quantitative image analysis, we developed random forest and deep learning-based pipelines for cell segmentation. The pipelines were integrated into the user-friendly open-source Aparecium software. Both image analysis methods were suitable for measuring fluorescence ligand saturation binding and kinetics as well as for screening binding affinities of unlabelled ligands.

A New Benzoxazole-Based Fluorescent Macrocyclic Chemosensor for Optical Detection of Zn2+ and Cd2+

Background: Benzoxazole-containing ligands find many applications both in medicinal chemistry, catalysis and fluorescence chemosensing. Benzoxazole-containing macrocycles could be therefore a good strategy to achieve stable and selective fluorescent complexes with suitable metal ions. In this work, the synthesis, binding, and photochemical properties of a new fluorescent ligand (L) are reported. L is a cyclophane macrocycle containing the 1,3-bis(benzo[d]oxazol-2-yl)phenyl (BBzB) fluorophore and an aliphatic tetra-amine chain to form the macrocyclic skeleton. Methods: Spectrophotometric and spectrofluorimetric measurements, 1H NMR analysis, and DFT calculations were performed. Results: L behaves as a PET-mediated chemosensor, being emissive at 390 nm at acidic pH and non-emissive at basic pH. The chemosensor is able to detect Zn2+ and Cd2+ in an aqueous medium (acetonitrile–water, 4:1 v/v) at neutral pH through a CHEF effect upon metal ion coordination. Paramagnetic metal ions (Cu2+) and heavy atoms (Pb2+, Hg2+) resulted in a quenching of fluorescence or very low emission. Conclusions: The new cyclophane macrocycle L was revealed to be a selective PET-regulated chemosensor for Zn2+ and Cd2+ in an aqueous medium, being able to bind up to two and one metal cations, respectively. The molecule showed a shifted emission towards the visible region compared to similar systems, suggesting a co-planar conformation of the aromatic fragment upon metal coordination. All these data are supported by both experimental measurements and theoretical calculations.

Spatial Expression Analysis of Odorant Binding Proteins in Both Sexes of the Aphid Parasitoid Aphidius gifuensis and Their Ligand Binding Properties.

In China, Aphidius gifuensis is one of the most common endoparasitoids of the green peach aphid Myzus persicae and grain aphid Sitobion miscanthi in the field. Insect odorant-binding proteins (OBPs) play vital roles in odor perception during feeding, host searching, mating and oviposition. In addition, some OBPs are involved in other physiological processes such as gustation and reproduction. In the present study, a comparative antennal transcriptomic analysis was applied between male and female A. gifuensis. The spatial expression patterns among antennae, heads, thoraxes, abdomens and legs of OBPs in both sexes were further profiled. Fifteen AgifOBPs were predicted, and 14 of them were identified by gene cloning, including 12 classic OBPs and 2 min-C OBPs. As expected, all OBPs were mainly expressed at high levels in antennae, heads or legs which are sensory organs and tissues. Finally, ligand binding properties of 2 OBPs (AgifOBP7 and AgifOBP9) were further evaluated. Female leg specifically expressed AgifOBP9 displays a broad and high binding property to aphid alarm pheromones, plant green volatiles and aphid sex pheromones (Ki < 10 μΜ). However, female leg specifically expressed AgifOBP7 displays poor affinity for all tested ligands except CAU-II-11 ((E)-3,7-dimethylocta-2,6-dien-1-yl-2-hydroxy-3-methoxybenzoate), a reported (E)-β-farnesene (EBF) analog with an exceptionally high binding affinity (Ki = 1.07 ± 0.08 μΜ). In summary, we reported the spatial expression pattern of the OBP repertoire in A. gifuensis, and further studied the binding properties of OBP7 and OBP9, which are mainly expressed in female legs, laying the foundation for the dissection of the contribution of OBPs to chemosensation in A. gifuensis.

Resonance Raman of the nitrite reductase action of heme‐copper oxidoreductases

Several themes have emerged in the studies of biological control of nitrite and nitric‐oxide chemistry.[1‐2] Apparently, evolution has clearly selected means by which to take advantage of the unique NO2 and NO chemistry, and a number of enzymes have evolved that are able to activate NO2/NO kinetically, and control their redox chemistry. In bacterial respiration the reduction of NO2/NO to N2O by terminal oxidoreductases supports the hypothesis of a common evolutionary origin of bacterial denitrification and aerobic respiration. Comparison of the enzymes responsible for the activation of NO in denitrification and bacterial respiration may provide the means to identify conserved structural features, which can be assumed to be involved in basic functions common to both classes of enzymes. The most commonly encountered metals in the biological reduction of NO2 to NO and subsequently to N2O are iron and copper in mononuclear, binuclear, and dinuclear centers. In P450Nor the electrons needed for the reduction of NO are directly transferred from NADH. This way, a single molecule of NO binds at the heme Fe, and addition of two electrons to heme Fe3+‐NO yields the two electron reduced species Fe2+‐N=O‐. A second NO moleculeattacks the N atom of the ferrous‐NO species to transiently yield hyponitrite (HONNO–), and thus the N‐N bond formation. Cleavage of the N‐O bond produces the ferric enzyme, N2O and H2O. Resonance Raman studies of the NO2‐ligand binding properties of heme‐copper oxidases will be presented.References. Discrete Ligand Binding and Electron Transfer Properties of ba3‐Cytochrome c Oxidase from Thermus thermophilus: Evolutionary Adaption to Low Oxygen and …C Koutsoupakis, T Soulimane, C. Varotsis Accounts of chemical research 52 (5), 1380‐139, 2019. The structure of a ferrous heme‐nitro species in the binuclear heme a3/CuBcenter of ba3‐cytochrome c oxidase as determined by resonance Raman spectroscopy A. Loullis, MR Noor, T Soulimane, E Pinakoulaki Chemical Communications 51 (2), 286‐289, 2015.

αα-hub coregulator structure and flexibility determine transcription factor binding and selection in regulatory interactomes

Formation of transcription factor (TF)–coregulator complexes is a key step in transcriptional regulation, with coregulators having essential functions as hub nodes in molecular networks. How specificity and selectivity are maintained in these nodes remain open questions. In this work, we addressed specificity in transcriptional networks using complexes formed between TFs and αα-hubs, which are defined by a common αα-hairpin secondary structure motif, as a model. Using NMR spectroscopy and binding thermodynamics, we analyzed the structure, dynamics, stability, and ligand-binding properties of the Arabidopsis thaliana RST domains from TAF4 and known binding partner RCD1, and the TAFH domain from human TAF4, allowing comparison across species, functions, and architectural contexts. While these αα-hubs shared the αα-hairpin motif, they differed in length and orientation of accessory helices as well as in their thermodynamic profiles of ligand binding. Whereas biologically relevant RCD1–ligand pairs displayed high affinity driven by enthalpy, TAF4–ligand interactions were entropy driven and exhibited less binding-induced structuring. We in addition identified a thermal unfolding state with a structured core for all three domains, although the temperature sensitivity differed. Thermal stability studies suggested that initial unfolding of the RCD1–RST domain localized around helix 1, lending this region structural malleability, while effects in TAF4–RST were more stochastic, suggesting variability in structural adaptability upon binding. Collectively, our results support a model in which hub structure, flexibility, and binding thermodynamics contribute to αα-hub–TF binding specificity, a finding of general relevance to the understanding of coregulator–ligand interactions and interactome sizes.

Machine Learning Approaches for Metalloproteins.

Metalloproteins are a family of proteins characterized by metal ion binding, whereby the presence of these ions confers key catalytic and ligand-binding properties. Due to their ubiquity among biological systems, researchers have made immense efforts to predict the structural and functional roles of metalloproteins. Ultimately, having a comprehensive understanding of metalloproteins will lead to tangible applications, such as designing potent inhibitors in drug discovery. Recently, there has been an acceleration in the number of studies applying machine learning to predict metalloprotein properties, primarily driven by the advent of more sophisticated machine learning algorithms. This review covers how machine learning tools have consolidated and expanded our comprehension of various aspects of metalloproteins (structure, function, stability, ligand-binding interactions, and inhibitors). Future avenues of exploration are also discussed.

Electrophoretic study of G-quadruplex aptamer interactions with different short single-strand complementary oligonucleotides

Aptamers are single-stranded nucleic acids, typically 20-80 nucleobases (nb) in length, which can bind different compounds with high affinity and selectively. Their ligand-binding properties can be attenuated by adding short complementary strands. These interactions open new opportunities for aptamer-based assays. Strong dependence between the length and electrophoretic mobility of short nucleic acids makes polyacrylamide gel electrophoresis a powerful tool for studying their complexes. The interactions between the 36 nb DNA G-quadruplex aptamer (5’-GAT-CGG-GTG-TGG-GTG-GCG-TAA-AGG-GAG-CAT-CGG-ACA-3’) specific to ochratoxin A and 9 complementary single-stranded DNA (ssDNA) were studied. The length of ssDNA varied from 5 to 9 nb. To maintain ligand-binding conformation of the aptamer, a high ionic strength buffer was used. The best resolution between the aptamer and its complex was provided for the gel with 15% monomer and a monomer/cross-linker ratio of 15:1. Bands of free aptamer and ssDNA were observed for all studied variants. If the ssDNA length was less than 9 nb, the position of the aptamer’s band remained unchanged, independent of the aptamer/ssDNA ratios, and additional bands did not appear. The longest ssDNA (5’-CGC-CAC-CCA-3’) did not lead to the appearance of a new band, but it slowed the aptamer’s migration depending on the ssDNA concentration. Under a 27-fold excess of the given ssDNA, the relative mobility of the aptamer band changed from 0.566 to 0.468. Thus, electrophoresis visualizes aptamer-ssDNA interactions and can be used in the development of aptamer-based analytical systems.

Therapeutic potential of opioid receptor heteromers in chronic pain and associated comorbidities.

Chronic pain affects 20% to 45% of the global population and is often associated with the development of anxio-depressive disorders. Treatment of this debilitating condition remains particularly challenging with opioids prescribed to alleviate moderate to severe pain. However, despite strong antinociceptive properties, numerous adverse effects limit opioid use in the clinic. Moreover, opioid misuse and abuse have become a major health concern worldwide. This prompted efforts to design original strategies that would efficiently and safely relieve pain. Targeting of opioid receptor heteromers is one of these. This review summarizes our current knowledge on the role of heteromers involving opioid receptors in the context of chronic pain and anxio-depressive comorbidities. It also examines how heteromerization in native tissue affects ligand binding, receptor signalling and trafficking properties. Finally, the therapeutic potential of ligands designed to specifically target opioid receptor heteromers is considered. LINKED ARTICLES: This article is part of a themed issue on Advances in Opioid Pharmacology at the Time of the Opioid Epidemic. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v180.7/issuetoc.

Binding of Hepatitis B Virus Pre-S1 Domain-Derived Synthetic Myristoylated Peptide to Scavenger Receptor Class B Type 1 with Differential Properties from Sodium Taurocholate Cotransporting Polypeptide.

(1) Background: The myristoylated pre-S1 peptide (Myr47) synthesized to mimic pre-S1 domain (2-48) in large (L) surface protein of hepatitis B virus (HBV) prevents HBV infection to hepatocytes by binding to sodium taurocholate cotransporting polypeptide (NTCP). We previously demonstrated that yeast-derived nanoparticles containing L protein (bio-nanocapsules: BNCs) bind scavenger receptor class B type 1 (SR-B1). In this study, we examined the binding of Mry47 to SR-B1. (2) Methods: The binding and endocytosis of fluorescence-labeled Myr47 to SR-B1 (and its mutants)-green fluorescence protein (GFP) fusion proteins expressed in HEK293T cells were analyzed using flow cytometry and laser scanning microscopy (LSM). Various ligand-binding properties were compared between SR-B1-GFP and NTCP-GFP. Furthermore, the binding of biotinylated Myr47 to SR-B1-GFP expressed on HEK293T cells was analyzed via pull-down assays using a crosslinker and streptavidin-conjugated beads. (3) Conclusions: SR-B1 bound not only Myr47 but also its myristoylated analog and BNCs, but failed to bind a peptide without myristoylation. However, NTCP only bound Myr47 among the ligands tested. Studies using SR-B1 mutants suggested that both BNCs and Myr47 bind to similar sites of SR-B1. Crosslinking studies indicated that Myr47 binds preferentially SR-B1 multimer than monomer in both HEK293T and HepG2 cells.

Development and Characterization of a Fluorescent Ligand for Leukotriene B4 Receptor 2 in Cells and Tissues.

The leukotriene B4 receptor 2 (BLT2) is a G-protein coupled receptor activated by 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT), which has been proposed as a promising therapeutic target for diabetic wound healing and gastrointestinal lesions. In this study, the rational design of a fluorescent probe based on the synthetic BLT2 agonist CAY10583 is described. The synthesis of several derivatives of CAY10583 coupled to fluorescein resulted in a traceable ligand suitable for different fluorescence-based techniques. An HTRF-based displacement assay (Tag-lite) on stably transfected CHO-K1 cells was developed to characterize binding properties of diverse BLT2 ligands. Highly specific binding to the BLT2 receptor was demonstrated in staining experiments on mouse skin tissue, and specific modulation of BLT2-induced cAMP signaling provided further evidence for receptor binding and ligand functionality. In conclusion, the fluorescent ligands developed in this study are suitable to investigate the pharmacology of BLT2 receptor ligands in a variety of assay systems.

Translocator Protein (18 kDa) Polymorphism (rs6971) in the Korean Population.

Background and PurposeThe expression of the 18-kDA mitochondrial translocator protein (TSPO) in the brain is an attractive target to study neuroinflammation. However, the binding properties of TSPO ligands are reportedly dependent on genetic polymorphism of the TSPO gene (rs6971). The objective of this study is to investigate the rs6971 gene polymorphism in the Korean population.MethodsWe performed genetic testing on 109 subjects including patients with mild cognitive impairment, Alzheimer’s disease (AD) dementia, non-AD dementia, and cognitively unimpaired participants. Magnetic resonance imaging scans and detailed neuropsychological tests were also performed, and 29 participants underwent 18F-DPA714 PET scans. Exon 4 of the TSPO gene containing the polymorphism rs6971 (Ala or Thr at position 147) was polymerase chain reaction amplified and sequenced using the Sanger method. The identified rs6971 genotype codes (C/C, C/T, or T/T) of the TSPO protein generated high-, mixed-, or low-affinity binding phenotypes (HABs, MABs, and LABs), respectively.ResultsWe found that 96.3% of the study subjects were HAB (105 out of 109 subjects), and 3.7% of the subjects were MAB (4 out of 109 subjects). 18F-DPA-714 PET scans showed nonspecific binding to the thalamus and brainstem, and increased tracer uptake throughout the cortex in cognitively impaired patients. The participant with the MAB polymorphism had a higher DPA714 signal throughout the cortex.ConclusionsThe majority of Koreans are HAB (aprox. 96%). Therefore, the polymorphism of the rs6971 gene would have a smaller impact on the availability of second-generation TSPO PET tracers.

Purification, molecular characterization and ligand binding properties of the major donkey seminal plasma protein DSP-1

Fibronectin type-II (FnII) family proteins are the major proteins in many mammalian species including bull, horse and pig. In the present study, a major FnII protein has been identified and isolated from donkey (Equus hemionus) seminal plasma, which we refer to as Donkey Seminal Plasma protein-1 (DSP-1). The amino acid sequence determined by mass spectrometry and computational modeling studies revealed that DSP-1 is homologous to other mammalian seminal plasma proteins, including bovine PDC-109 (also known as BSP-A1/A2) and equine HSP-1/2. High-resolution LC-MS analysis indicated that the protein is heterogeneously glycosylated and also contains multiple acetylations, occurring in the attached glycans. Structural and thermal stability studies on DSP-1 employing CD spectroscopy and differential scanning calorimetry showed that the protein unfolds at ~43 °C and binding to phosphorylcholine (PrC) – the head group moiety of choline phospholipids – increases its thermal stability. Intrinsic fluorescence titrations revealed that DSP-1 recognizes lyso-phosphatidylcholine with over 100-fold higher affinity than PrC. Further, interaction of DSP-1 with erythrocytes, a model cell membrane, revealed that DSP-1 binding is mediated by a specific interaction with choline phospholipids and results in membrane perturbation, suggesting that binding of this protein to sperm plasma membrane could be physiologically significant.

Targeting Human Osteoarthritic Chondrocytes with Ligand Directed Bacteriophage-Based Particles.

Osteoarthritis (OA) is a degenerative joint disease characterized by progressive deterioration and loss of articular cartilage. There is currently no treatment to reverse the onset of OA. Thus, we developed a targeted delivery strategy to transfer genes into primary human chondrocytes as a proof-of-concept study. We displayed a chondrocyte-affinity peptide (CAP) on the pIII minor coat protein of the M13 filamentous bacteriophage (phage)-based particle carrying a mammalian transgene cassette under cytomegalovirus CMV promoter and inverted terminal repeats (ITRs) cis elements of adeno-associated virus serotype 2 (AAV-2). Primary human articular chondrocytes (HACs) were used as an in vitro model, and the selectivity and binding properties of the CAP ligand in relation to the pathogenic conditions of HACs were characterized. We found that the CAP ligand is highly selective toward pathogenic HACs. Furthermore, the stability, cytotoxicity, and gene delivery efficacy of the CAP-displaying phage (CAP.Phage) were evaluated. We found that the phage particle is stable under a wide range of temperatures and pH values, while showing no cytotoxicity to HACs. Importantly, the CAP.Phage particle, carrying a secreted luciferase (Lucia) reporter gene, efficiently and selectively delivered transgene expression to HACs. In summary, it was found that the CAP ligand preferably binds to pathogenic chondrocytes, and the CAP.Phage particle successfully targets and delivers transgene to HACs.

Biophysical and functional characterization of the human TAS1R2 sweet taste receptor overexpressed in a HEK293S inducible cell line

Sweet taste perception is mediated by a heterodimeric receptor formed by the assembly of the TAS1R2 and TAS1R3 subunits. TAS1R2 and TAS1R3 are class C G-protein-coupled receptors whose members share a common topology, including a large extracellular N-terminal domain (NTD) linked to a seven transmembrane domain (TMD) by a cysteine-rich domain. TAS1R2-NTD contains the primary binding site for sweet compounds, including natural sugars and high-potency sweeteners, whereas the TAS1R2-TMD has been shown to bind a limited number of sweet tasting compounds. To understand the molecular mechanisms governing receptor–ligand interactions, we overexpressed the human TAS1R2 (hTAS1R2) in a stable tetracycline-inducible HEK293S cell line and purified the detergent-solubilized receptor. Circular dichroism spectroscopic studies revealed that hTAS1R2 was properly folded with evidence of secondary structures. Using size exclusion chromatography coupled to light scattering, we found that the hTAS1R2 subunit is a dimer. Ligand binding properties were quantified by intrinsic tryptophan fluorescence. Due to technical limitations, natural sugars have not been tested. However, we showed that hTAS1R2 is capable of binding high potency sweeteners with Kd values that are in agreement with physiological detection. This study offers a new experimental strategy to identify new sweeteners or taste modulators that act on the hTAS1R2 and is a prerequisite for structural query and biophysical studies.

Subcellular Localization Defects Characterize Ribose-Binding Mutant Proteins with New Ligand Properties in Escherichia coli.

Periplasmic binding proteins have been previously proclaimed as a general scaffold to design sensor proteins with new recognition specificities for nonnatural compounds. Such proteins can be integrated in bacterial bioreporter chassis with hybrid chemoreceptors to produce a concentration-dependent signal after ligand binding to the sensor cell. However, computationally designed new ligand-binding properties ignore the more general properties of periplasmic binding proteins, such as their periplasmic translocation, dynamic transition of open and closed forms, and interactions with membrane receptors. In order to better understand the roles of such general properties in periplasmic signaling behavior, we studied the subcellular localization of ribose-binding protein (RbsB) in Escherichia coli in comparison to a recently evolved set of mutants designed to bind 1,3-cyclohexanediol. As proxies for localization, we calibrated and deployed C-terminal end mCherry fluorescent protein fusions. Whereas RbsB-mCherry coherently localized to the periplasmic space and accumulated in (periplasmic) polar regions depending on chemoreceptor availability, mutant RbsB-mCherry expression resulted in high fluorescence cell-to-cell variability. This resulted in higher proportions of cells devoid of clear polar foci and of cells with multiple fluorescent foci elsewhere, suggesting poorer translocation, periplasmic autoaggregation, and mislocalization. Analysis of RbsB mutants and mutant libraries at different stages of directed evolution suggested overall improvement to more RbsB-wild-type-like characteristics, which was corroborated by structure predictions. Our results show that defects in periplasmic localization of mutant RbsB proteins partly explain their poor sensing performance. Future efforts should be directed to predicting or selecting secondary mutations outside computationally designed binding pockets, taking folding, translocation, and receptor interactions into account. IMPORTANCE Biosensor engineering relies on transcription factors or signaling proteins to provide the actual sensory functions for the target chemicals. Since for many compounds there are no natural sensory proteins, there is a general interest in methods that could unlock routes to obtaining new ligand-binding properties. Bacterial periplasmic binding proteins (PBPs) form an interesting family of proteins to explore for this purpose, because there is a large natural variety suggesting evolutionary trajectories to bind new ligands. PBPs are conserved and amenable to accurate computational binding pocket predictions. However, studying ribose-binding protein in Escherichia coli, we discovered that designed variants have defects in their proper localization in the cell, which can impair appropriate sensor signaling. This indicates that functional sensing capacity of PBPs cannot be obtained solely through computational design of the ligand-binding pocket but must take other properties of the protein into account, which are currently very difficult to predict.

The structural basis for high affinity binding of α1-acid glycoprotein to the potent antitumor compound UCN-01

The α1-acid glycoprotein (AGP) is an abundant blood plasma protein with important immunomodulatory functions coupled to endogenous and exogenous ligand-binding properties. Its affinity for many drug-like structures, however, means AGP can have a significant effect on the pharmokinetics and pharmacodynamics of numerous small molecule therapeutics. Staurosporine, and its hydroxylated forms UCN-01 and UCN-02, are kinase inhibitors that have been investigated at length as antitumour compounds. Despite their potency, these compounds display poor pharmokinetics due to binding to both AGP variants, AGP1 and AGP2. The recent renewed interest in UCN-01 as a cytostatic protective agent prompted us to solve the structure of the AGP2–UCN-01 complex by X-ray crystallography, revealing for the first time the precise binding mode of UCN-01. The solution NMR suggests AGP2 undergoes a significant conformational change upon ligand binding, but also that it uses a common set of sidechains with which it captures key groups of UCN-01 and other small molecule ligands. We anticipate that this structure and the supporting NMR data will facilitate rational redesign of small molecules that could evade AGP and therefore improve tissue distribution.

The Influence of AA29504 on GABAA Receptor Ligand Binding Properties and Its Implications on Subtype Selectivity

The unique pharmacological properties of δ-containing γ-aminobutyric acid type A receptors (δ-GABAARs) make them an attractive target for selective and persistent modulation of neuronal excitability. However, the availability of selective modulators targeting δ-GABAARs remains limited. AA29504 ([2-amino-4-(2,4,6-trimethylbenzylamino)-phenyl]-carbamic acid ethyl ester), an analog of K+ channel opener retigabine, acts as an agonist and a positive allosteric modulator (Ago-PAM) of δ-GABAARs. Based on electrophysiological studies using recombinant receptors, AA29504 was found to be a more potent and effective agonist in δ-GABAARs than in γ2-GABAARs. In comparison, AA29504 positively modulated the activity of recombinant δ-GABAARs more effectively than γ2-GABAARs, with no significant differences in potency. The impact of AA29504's efficacy- and potency-associated GABAAR subtype selectivity on radioligand binding properties remain unexplored. Using [3H]4'-ethynyl-4-n-propylbicycloorthobenzoate ([3H]EBOB) binding assay, we found no difference in the modulatory potency of AA29504 on GABA- and THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol)-induced responses between native forebrain GABAARs of wild type and δ knock-out mice. In recombinant receptors expressed in HEK293 cells, AA29504 showed higher efficacy on δ- than γ2-GABAARs in the GABA-independent displacement of [3H]EBOB binding. Interestingly, AA29504 showed a concentration-dependent stimulation of [3H]muscimol binding to γ2-GABAARs, which was absent in δ-GABAARs. This was explained by AA29504 shifting the low-affinity γ2-GABAAR towards a higher affinity desensitized state, thereby rising new sites capable of binding GABAAR agonists with low nanomolar affinity. Hence, the potential of AA29504 to act as a desensitization-modifying allosteric modulator of γ2-GABAARs deserves further investigation for its promising influence on shaping efficacy, duration and plasticity of GABAAR synaptic responses.