Specific Ligand Binding Research Articles

Structural Insights into the Specificity of Ligand Binding and Coactivator Assembly by Estrogen-Related Receptor β

Estrogen-related receptor β (ERRβ) is a nuclear receptor critical for many biological processes. Despite the biological and pharmaceutical importance of ERRβ, deciphering the structure of ERRβ has been hampered by the difficulties in obtaining a pure and stable protein for structural studies. In fact, the ERRβ ligand-binding domain remains the last unsolved ERR structure and also one of only a few unknown nuclear receptor structures. Here, we report the identification of a critical single-residue mutation resulted in robust solubility and stability of an active ERRβ ligand-binding domain, thereby providing a protein tool enabling the first probe into the biochemical and structural studies of this important receptor. The crystal structure reveals key structural features that have enabled the integration of the molecular determinants of signals transduced across the ligand binding and coregulator recruitment by all three ERR subtypes, which also provides a framework for the rational design of selective and potent ligands for the treatment of various ERR-mediated diseases.

That's a Wrap! Molecular Drivers Governing Neuronal Nogo Receptor-Dependent Myelin Plasticity and Integrity.

Myelin is a dynamic membrane that is important for coordinating the fast propagation of action potentials along small or large caliber axons (0.1–10 μm) some of which extend the entire length of the spinal cord. Due to the heterogeneity of electrical and energy demands of the variable neuronal populations, the axo-myelinic and axo-glial interactions that regulate the biophysical properties of myelinated axons also vary in terms of molecular interactions at the membrane interfaces. An important topic of debate in neuroscience is how myelin is maintained and modified under neuronal control and how disruption of this control (due to disease or injury) can initiate and/or propagate neurodegeneration. One of the key molecular signaling cascades that have been investigated in the context of neural injury over the past two decades involves the myelin-associated inhibitory factors (MAIFs) that interact with Nogo receptor 1 (NgR1). Chief among the MAIF superfamily of molecules is a reticulon family protein, Nogo-A, that is established as a potent inhibitor of neurite sprouting and axon regeneration. However, an understated role for NgR1 is its ability to control axo-myelin interactions and Nogo-A specific ligand binding. These interactions may occur at axo-dendritic and axo-glial synapses regulating their functional and dynamic membrane domains. The current review provides a comprehensive analysis of how neuronal NgR1 can regulate myelin thickness and plasticity under normal and disease conditions. Specifically, we discuss how NgR1 plays an important role in regulating paranodal and juxtaparanodal domains through specific signal transduction cascades that are important for microdomain molecular architecture and action potential propagation. Potential therapeutics designed to target NgR1-dependent signaling during disease are being developed in animal models since interference with the involvement of the receptor may facilitate neurological recovery. Hence, the regulatory role played by NgR1 in the axo-myelinic interface is an important research field of clinical significance that requires comprehensive investigation.

Functional reprogramming of Candida glabrata epithelial adhesins: the role of conserved and variable structural motifs in ligand binding

For host-cell interaction, the human fungal pathogen Candida glabrata harbors a large family of more than 20 cell wall-attached epithelial adhesins (Epas). Epa family members are lectins with binding pockets containing several conserved and variable structural hot spots, which were implicated in mediating functional diversity. In this study, we have performed an elaborate structure-based mutational analysis of numerous Epa paralogs to generally determine the role of diverse structural hot spots in conferring host cell binding and ligand binding specificity. Our study reveals that several conserved structural motifs contribute to efficient host cell binding. Moreover, our directed motif exchange experiments reveal that the variable loop CBL2 is key for programming ligand binding specificity, albeit with limited predictability. In contrast, we find that the variable loop L1 affects host cell binding without significantly influencing the specificity of ligand binding. Our data strongly suggest that variation of numerous structural hot spots in the ligand binding pocket of Epa proteins is a main driver of their functional diversification and evolution.

ApoER2: Functional Tuning Through Splicing.

Alternative splicing occurs in over 95% of protein-coding genes and contributes to the diversity of the human proteome. Apolipoprotein E receptor 2 (apoER2) is a critical modulator of neuronal development and synaptic plasticity in the brain and is enriched in cassette exon splicing events, in which functional exons are excluded from the final transcript. These alternative splicing events affect apoER2 function, as individual apoER2 exons tend to encode distinct protein functional domains. Although several apoER2 splice variants have been characterized, much work remains to understand how apoER2 splicing events modulate distinct apoER2 activities, including ligand binding specificity, synapse formation and plasticity. Additionally, little is known about how apoER2 splicing events are regulated. Often, alternative splicing events are regulated through the combinatorial action of RNA-binding proteins and other epigenetic mechanisms, however, the regulatory pathways corresponding to each specific exon are unknown in most cases. In this mini-review, we describe the structure of apoER2, highlight the unique functions of known isoforms, discuss what is currently known about the regulation of apoER2 splicing by RNA-binding proteins and pose new questions that will further our understanding of apoER2 splicing complexity.

Microglia: Agents of the CNS Pro-Inflammatory Response.

The pro-inflammatory immune response driven by microglia is a key contributor to the pathogenesis of several neurodegenerative diseases. Though the research of microglia spans over a century, the last two decades have increased our understanding exponentially. Here, we discuss the phenotypic transformation from homeostatic microglia towards reactive microglia, initiated by specific ligand binding to pattern recognition receptors including toll-like receptor-4 (TLR4) or triggering receptors expressed on myeloid cells-2 (TREM2), as well as pro-inflammatory signaling pathways triggered such as the caspase-mediated immune response. Additionally, new research disciplines such as epigenetics and immunometabolism have provided us with a more holistic view of how changes in DNA methylation, microRNAs, and the metabolome may influence the pro-inflammatory response. This review aimed to discuss our current knowledge of pro-inflammatory microglia from different angles, including recent research highlights such as the role of exosomes in spreading neuroinflammation and emerging techniques in microglia research including positron emission tomography (PET) scanning and the use of human microglia generated from induced pluripotent stem cells (iPSCs). Finally, we also discuss current thoughts on the impact of pro-inflammatory microglia in neurodegenerative diseases.

Development of a method to screen and isolate lipoxidase inhibitors from Radix Saposhnikoviae via ultrafiltration liquid chromatography combined with metablism in vitro.

Radix Saposhnikoviae is an excellent source of chromones, the consumption of which has been shown to positively affect human health, such as anti-inflammatory and antioxidant activities in vivo, and it also has been used to treat headache, inflammation, and cancer as a herbal medicine or functional soup. Isolation and screening of lipoxidase (LOX) inhibitors from Radix Saposhnikoviae. As a result, the metabolism of the active ingredients in cytochrome P450 (CYP450) in vitro was identified. Screen and identify the LOX inhibitors from Radix Saposhnikoviae by ultrafiltration-liquid chromatography-mass spectrometry (UF-LC-MS). Then take the isolation and purification of the inhibitors by high-speed counter-current chromatography (HSCCC) and semi-preparative high-performance liquid chromatography (HPLC). Furthermore, the active ingredients were used to study the metabolism in vitro in CYP450. Subsequently, the specific binding ligands, prim-O-glucosylcimifugin, cimifugin, and 5-O-methylvisamminol (with purities of 95.28, 92.33%, and 97.89, respectively) were separated by HSCCC. Semi-preparative HPLC was applied to separate the four major compounds in Radix Saposhnikoviae and identify them as prim-O-glucosylcimifugin, 5-O-methylvisamminol, sec-O-glucosylhamaudol, and hamaudol, which were obtained with relatively high purity. Furthermore, the three chromone possible metabolism pathways were obtained. The results demonstrated that UF-LC-MS combined with HSCCC and semi-preparative HPLC is not only a powerful tool for screening and isolating LOX inhibitors in complex samples, but also a useful platform for discovering bioactive compounds for anti-inflammatory activity. This study provides information regarding the active ingredients of chromones in Radix Saposhnikoviae and a framework for anti-inflammatory activity treatment.

ADP binding by the Culex quinquefasciatus mosquito D7 salivary protein enhances blood feeding on mammals

During blood-feeding, mosquito saliva is injected into the skin to facilitate blood meal acquisition. D7 proteins are among the most abundant components of the mosquito saliva. Here we report the ligand binding specificity and physiological relevance of two D7 long proteins from Culex quinquefasciatus mosquito, the vector of filaria parasites or West Nile viruses. CxD7L2 binds biogenic amines and eicosanoids. CxD7L1 exhibits high affinity for ADP and ATP, a binding capacity not reported in any D7. We solve the crystal structure of CxD7L1 in complex with ADP to 1.97 Å resolution. The binding pocket lies between the two protein domains, whereas all known D7s bind ligands either within the N- or the C-terminal domains. We demonstrate that these proteins inhibit hemostasis in ex vivo and in vivo experiments. Our results suggest that the ADP-binding function acquired by CxD7L1 evolved to enhance blood-feeding in mammals, where ADP plays a key role in platelet aggregation.

Immobilized DNA Switch Modulated by Intermolecular Interactions

Conventional DNA constructs with integrated aptamer motifs usually undergo “global-scale” conformational and associated conductivity switches in response to specific ligand binding, which can be ex...

Quicker, deeper and stronger imaging: A review of tumor-targeted, near-infrared fluorescent dyes for fluorescence guided surgery in the preclinical and clinical stages

Cancer is a public health problem and the main cause of human mortality and morbidity worldwide. Complete removal of tumors and metastatic lymph nodes in surgery is significantly beneficial for the prognosis of patients. Tumor-targeted, near-infrared fluorescent (NIRF) imaging is an emerging field of real-time intraoperative cancer imaging based on tumor-targeted NIRF dyes. Targeted NIRF dyes contain NIRF fluorophores and specific binding ligands such as antibodies, peptides and small molecules. The present article reviews recently updated tumor-targeted NIRF dyes for the molecular imaging of malignant tumors in the preclinical stage and clinical trials. The strengths and challenges of NIRF agents with tumor-targeting ability are also summarized. Smaller ligands, near infrared II dyes, dual-modality dyes and activatable dyes may contribute to quicker, deeper, stronger imaging in the nearest future. In this review, we highlighted tumor-targeted NIRF dyes for fluorescence-guided surgery and their potential clinical translation.

Complement activation by C-reactive protein is essential for protection against pneumococcal infection

Abstract C-reactive protein (CRP), a component of the innate immune system, is an antimicrobial plasma protein. The primary ligand-binding specificity of CRP is for phosphocholine-containing substances and ligand-complexed human CRP activates the complement system in both human and mouse sera. It has been shown that human CRP protects mice against infection with lethal doses of Streptococcus pneumoniae; the protection is due to CRP-dependent decrease in bacteremia and increase in survival time. In this study, we tested a decades old hypothesis that the complement-activating function of ligand-complexed CRP contributes to protection of mice against pneumococcal infection. We employed site-directed mutagenesis of CRP, guided by its three-dimensional structure, and identified a mutant H38R which, unlike wild-type CRP, did not activate C3 in both human and mouse sera. The calcium-dependent binding to phosphocholine on pneumococci, the half-life in mouse circulation, and the overall pentameric structure of H38R mutant CRP were similar to that of wild-type CRP. Employing a murine model of pneumococcal infection, we found that passively administered H38R mutant CRP did not protect mice from infection. Infected mice injected with H38R mutant CRP showed no reduction in bacteremia and did not survive longer, as opposed to infected mice receiving wild-type CRP. Thus, the hypothesis that complement activation by ligand-complexed CRP is an antimicrobial effector function is true. We conclude that complement activation by ligand-complexed CRP is essential for CRP-mediated protection against pneumococcal infection. (NIH AI117730)

Screening of protein-ligand interactions under crude conditions by native mass spectrometry.

A convenient analytical system for protein-ligand interactions under crude conditions was developed using native mass spectrometry (MS). As a model protein, Escherichia coli (E. coli) dihydrofolate reductase (DHFR) with and without a histidine tag was used for the study. First, overexpressed DHFR with a His-tag was roughly purified with a Ni-sepharose resin and subjected to native mass spectrometry with or without incubation with an inhibitor, Methotrexate (MTX). Even only with the minimum cleanup by the Ni-sepharose resin, intact ions of DHFR-nicotinamide adenine dinucleotide phosphate (NADPH) and DHFR-NADPH-ligand complexes were successfully observed. By optimizing the preparation procedures of the crude sample for native MS, e.g., avoiding sonication for cell lysis, we successfully observed intact ions of the specific DHFR-NADPH-MTX ternary complex starting with cultivation of E. coli in ≤ 25mL medium. When the crude DHFR sample was mixed with two, four, or eight candidate compounds, only ions of the specific protein-ligand complex were observed. This indicates that the present system can be used as a rapid and convenient method for the rough determination of binding of specific ligands to the target protein without the time-consuming purification of protein samples. Moreover, it is important to rapidly determine specific interactions with target proteins under conditions similar to those in "real" biological systems. Graphical abstract.

Fuzzy Interactions Between 14‐3‐3ζ And Tau Regulate Tau Aggregation

The microtubule associated protein tau is vital for elongation and stability of microtubules in the central nervous system. However, aberrant accumulation of tau amyloid fibrils in neurons is a character of a neurodegenerative pathology called Alzheimer’s disease. Extensive studies have revealed that several factors promote aggregation of tau, including mutations, polyanions, phosphorylation, and interactions with other proteins. Previous studies have shown that the conserved scaffolding protein family 14‐3‐3 proteins interact with tau in a phosphorylation dependent manner and 14‐3‐3 proteins promote tau aggregation. Crystal structure of 14‐3‐3/tau complex revealed that phosphorylated tau segments bind to the ligand binding grooves of 14‐3‐3 dimer. Consequently, it is promising that 14‐3‐3 specific binding ligands will reduce/inhibit tau aggregation. Indeed, rational designed inhibitors of 14‐3‐3/tau interaction have been found to reduce binding of PKA‐phosphorylated tau protein to 14‐3‐3ζ. Although thermodynamics characterization showed that non‐phosphorylated tau segments bind to 14‐3‐3 much weaker than phosphorylated tau segments, 14‐3‐3ζ also promotes non‐phosphorylated tau aggregation. In this work, we investigated the interactions between tau K18 construct and 14‐3‐3ζ. We found that non‐phosphorylated and PKA‐phosphorylated tau proteins showed similar aggregating propensity in the presence of 14‐3‐3ζ. Replacing Ser324 with Ala had little effect of tau aggregation mediated by 14‐3‐3ζ protein. We further saturated the 14‐3‐3ζ ligand binding grooves by a high affinity peptide R18 and found that the presence of R18 could not reduce tau aggregation. Molecular dynamics simulations showed that tau K18 interact with 14‐3‐3ζ via extensive fuzzy interactions. Our results suggest that besides binding to the canonical ligand binding groove, tau also interacts with 14‐3‐3ζ via nonspecific interactions which are responsible for tau aggregation and cannot be inhibited by 14‐3‐3ζ specific inhibitors.Support or Funding InformationThis work was supported by Hubei University of Technology

NELL2-Robo3 complex structure reveals mechanisms of receptor activation for axon guidance

Axon pathfinding is critical for nervous system development, and it is orchestrated by molecular cues that activate receptors on the axonal growth cone. Robo family receptors bind Slit guidance cues to mediate axon repulsion. In mammals, the divergent family member Robo3 does not bind Slits, but instead signals axon repulsion from its own ligand, NELL2. Conversely, canonical Robos do not mediate NELL2 signaling. Here, we present the structures of NELL-Robo3 complexes, identifying a mode of ligand engagement for Robos that is orthogonal to Slit binding. We elucidate the structural basis for differential binding between NELL and Robo family members and show that NELL2 repulsive activity is a function of its Robo3 affinity and is enhanced by ligand trimerization. Our results reveal a mechanism of oligomerization-induced Robo activation for axon guidance and shed light on Robo family member ligand binding specificity, conformational variability, divergent modes of signaling, and evolution.

Structural basis of Gs and Gi recognition by the human glucagon receptor.

Class B G protein-coupled receptors, an important class of therapeutic targets, signal mainly through the Gs class of heterotrimeric G proteins, although they do display some promiscuity in G protein binding. Using cryo-electron microscopy, we determined the structures of the human glucagon receptor (GCGR) bound to glucagon and distinct classes of heterotrimeric G proteins, Gs or Gi1 These two structures adopt a similar open binding cavity to accommodate Gs and Gi1 The Gs binding selectivity of GCGR is explained by a larger interaction interface, but there are specific interactions that affect Gi more than Gs binding. Conformational differences in the receptor intracellular loops were found to be key selectivity determinants. These distinctions in transducer engagement were supported by mutagenesis and functional studies.

Choosing a partner that fits

Structural Biology G protein–coupled receptors (GPCRs) are responsible for transducing diverse signals from outside to inside cells. This process requires specificity both in ligand binding to GPCRs and in coupling between GPCRs and their intracellular partners, G proteins. Qiao et al. determined the structure of the human glucagon receptor (GCGR), a type B GPCR, bound to glucagon and one of two heterotrimeric G proteins, Gs or Gi1. GCGR signals mainly through Gs, and the structures provide a basis for this specificity. Conformational changes in GCGR, relative to the inactive state, create a binding cavity for the G proteins. The pocket is opened sufficiently to accommodate a bulky binding motif in Gs. Gi1 can still bind but the pocket does not close around it, so there is a smaller interaction interface. Science , this issue p. [1346][1] [1]: /lookup/doi/10.1126/science.aaz5346

The Identification of Metal Ion Ligand-Binding Residues by Adding the Reclassified Relative Solvent Accessibility.

Many proteins realize their special functions by binding with specific metal ion ligands during a cell’s life cycle. The ability to correctly identify metal ion ligand-binding residues is valuable for the human health and the design of molecular drug. Precisely identifying these residues, however, remains challenging work. We have presented an improved computational approach for predicting the binding residues of 10 metal ion ligands (Zn2+, Cu2+, Fe2+, Fe3+, Co2+, Ca2+, Mg2+, Mn2+, Na+, and K+) by adding reclassified relative solvent accessibility (RSA). The best accuracy of fivefold cross-validation was higher than 77.9%, which was about 16% higher than the previous result on the same dataset. It was found that different reclassification of the RSA information can make different contributions to the identification of specific ligand binding residues. Our study has provided an additional understanding of the effect of the RSA on the identification of metal ion ligand binding residues.

Efficient Separation of Nucleic Acids with Different Secondary Structures by Metal-Organic Frameworks.

We report the use of metal-organic frameworks (MOFs) for the selective separation of nucleic acids (DNA and RNA) with different secondary structures through size, shape, length, and capability of conformational transition. Three MOFs with precisely controlled pore environments, Co-IRMOF-74-II, -III, and -IV, composed of Co2+ and organic linkers (II, III, and IV), respectively, were used for the inclusion of nucleic acid into their pores from the solution. This was proven to be a spontaneous process from disordered free state to restricted ordered state via circular dichroism (CD) spectroscopy. Three critical factors were identified for their inclusion: (1) size selection induced by steric hindrance, (2) conformation transition energy selection induced by stability, and (3) molecular weight selection. These selection rules were used to extract nucleic acids with flexible and unstable secondary structures from complex mixtures of multiple nucleic acids, leaving those with rigid and stable secondary structures in the mother liquor. This provides the possibility to separate and enrich nucleic acids in bulk through their different structure feature, which is highly desirable in genome-wide structural measurement of nucleic acids. Unlike methods that rely on specific binding antibodies or ligand, this MOF method is capable of selecting all kinds of nucleic acids with similar secondary structure features; therefore, it is suitable for the handling of a large variety and quantity of nucleic acids at the same time. This method also has the potential to gather information about the folding stability of biomolecules with secondary structures.

Cryo-EM structures of PAC1 receptor reveal ligand binding mechanism

The pituitary adenylate cyclase-activating polypeptide type I receptor (PAC1R) belongs to the secretin receptor family and is widely distributed in the central neural system and peripheral organs. Abnormal activation of the receptor mediates trigeminovascular activation and sensitization, which is highly related to migraine, making PAC1R a potential therapeutic target. Elucidation of PAC1R activation mechanism would benefit discovery of therapeutic drugs for neuronal disorders. PAC1R activity is governed by pituitary adenylate cyclase-activating polypeptide (PACAP), known as a major vasodilator neuropeptide, and maxadilan, a native peptide from the sand fly, which is also capable of activating the receptor with similar potency. These peptide ligands have divergent sequences yet initiate convergent PAC1R activity. It is of interest to understand the mechanism of PAC1R ligand recognition and receptor activity regulation through structural biology. Here we report two near-atomic resolution cryo-EM structures of PAC1R activated by PACAP38 or maxadilan, providing structural insights into two distinct ligand binding modes. The structures illustrate flexibility of the extracellular domain (ECD) for ligands with distinct conformations, where ECD accommodates ligands in different orientations while extracellular loop 1 (ECL1) protrudes to further anchor the ligand bound in the orthosteric site. By structure-guided molecular modeling and mutagenesis, we tested residues in the ligand-binding pockets and identified clusters of residues that are critical for receptor activity. The structures reported here for the first time elucidate the mechanism of specificity and flexibility of ligand recognition and binding for PAC1R, and provide insights toward the design of therapeutic molecules targeting PAC1R.

TLR7 and TLR8 Differentially Activate the IRF and NF-κB Pathways in Specific Cell Types to Promote Inflammation.

TLR7 and TLR8 are pattern recognition receptors that reside in the endosome and are activated by ssRNA molecules. TLR7 and TLR8 are normally part of the antiviral defense response, but they have also been implicated as drivers of autoimmune diseases such as lupus. The receptors have slightly different ligand-binding specificities and cellular expression patterns that suggest they have nonredundant specialized roles. How the roles of TLR7 and TLR8 differ may be determined by which cell types express each TLR and how the cells respond to activation of each receptor. To provide a better understanding of the effects of TLR7/8 activation, we have characterized changes induced by TLR-specific agonists in different human immune cell types and defined which responses are a direct consequence of TLR7 or TLR8 activation and which are secondary responses driven by type I IFN or cytokines produced subsequent to the primary response. Using cell sorting, gene expression analysis, and intracellular cytokine staining, we have found that the IFN regulatory factor (IRF) and NF-κB pathways are differentially activated downstream of the TLRs in various cell types. Studies with an anti-IFNAR Ab in human cells and lupus mice showed that inhibiting IFN activity can block secondary IFN-induced gene expression changes downstream of TLR7/8 activation, but not NF-κB-regulated genes induced directly by TLR7/8 activation at earlier timepoints. In summary, these results elucidate the different roles TLR7 and TLR8 play in immunity and inform strategies for potential treatment of autoimmune diseases driven by TLR7/8 activation.

Molecular Basis for Hormone Recognition and Activation of Corticotropin-Releasing Factor Receptors.

Corticotropin-releasing factor (CRF) and the three related peptides urocortins 1-3 (UCN1-UCN3) are endocrine hormones that control the stress responses by activating CRF1R and CRF2R, two members of class B G-protein-coupled receptors (GPCRs). Here, we present two cryoelectron microscopy (cryo-EM) structures of UCN1-bound CRF1R and CRF2R with the stimulatory G protein. In both structures, UCN1 adopts a single straight helix with its N terminus dipped into the receptor transmembrane bundle. Although the peptide-binding residues in CRF1R and CRF2R are different from other members of class B GPCRs, the residues involved in receptor activation and G protein coupling are conserved. In addition, both structures reveal bound cholesterol molecules to the receptor transmembrane helices. Our structures define the basis of ligand-binding specificity in the CRF receptor-hormone system, establish a common mechanism of class B GPCR activation and G protein coupling, and provide a paradigm for studying membrane protein-lipid interactions for class B GPCRs.