Phosphorylation of cytosolic hPGK1 affects protein stability and ligand binding: implications for its subcellular targeting in cancer.

Human phosphoglycerate kinase 1(hPGK1) is a key glycolytic enzyme that regulates the balance between ADP and ATP concentrations inside the cell. Phosphorylation of hPGK1 at S203 and S256 has been associated with enzyme import from the cytosol to the mitochondria and the nucleus respectively. These changes in subcellular locations drive tumorigenesis and are likely associated with site-specific changes in protein stability. In this work, we investigate the effects of site-specific phosphorylation on thermal and kinetic stability and protein structural dynamics by hydrogen-deuterium exchange (HDX) and molecular dynamics (MD) simulations. We also investigate the binding of 3-phosphoglycerate and Mg-ADP using these approaches. We show that the phosphomimetic mutation S256D reduces hPGK1 kinetic stability by 50-fold, with no effect of the mutation S203D. Calorimetric studies of ligand binding show a large decrease in affinity for Mg-ADP in the S256D variant, whereas Mg-ADP binding to the WT and S203D can be accurately investigated using protein kinetic stability and binding thermodynamic models. HDX and MD simulations confirmed the destabilization caused by the mutation S256D (with some long-range effects on stability) and its reduced affinity for Mg-ADP due to the strong destabilization of its binding site (particularly in the apo-state). Our research provides evidence suggesting that modifications in protein stability could potentially enhance the translocation of hPGK1 to the nucleus in cancer. While the structural and energetic basis of its mitochondrial import remain unknown.

Collagen-targeted Protein Nanomicelles for the Imaging of Non-Alcoholic Steatohepatitis.

In vivo molecular imaging tools hold immense potential to drive transformative breakthroughs by enabling researchers to visualize cellular and molecular interactions in real-time and/or at high resolution. These advancements will facilitate a deeper understanding of fundamental biological processes and their dysregulation in disease states. Here, we develop and characterize a self-assembling protein nanomicelle called collagen type I binding - thermoresponsive assembled protein (Col1-TRAP) that binds tightly to type I collagen in vitro with nanomolar affinity. For ex vivo visualization, Col1-TRAP is labeled with a near-infrared fluorescent dye (NIR-Col1-TRAP). Both Col1-TRAP and NIR-Col1-TRAP display approximately a 3.8-fold greater binding to type I collagen compared to TRAP when measured by surface plasmon resonance (SPR). We present a proof-of-concept study using NIR-Col1-TRAP to detect fibrotic type I collagen deposition ex vivo in the livers of mice with non-alcoholic steatohepatitis (NASH). We show that NIR-Col1-TRAP demonstrates significantly decreased plasma recirculation time as well as increased liver accumulation in the NASH mice compared to mice without disease over 4 hours. As a result, NIR-Col1-TRAP shows potential as an imaging probe for NASH with in vivo targeting performance after injection in mice. STATEMENT OF SIGNIFICANCE: : Direct molecular imaging of fibrosis in NASH patients enables the diagnosis and monitoring of disease progression with greater specificity and resolution than do elastography-based methods or blood tests. In addition, protein-based imaging probes are more advantageous than alternatives due to their biodegradability and scalable biosynthesis. With the aid of computational modeling, we have designed a self-assembled protein micelle that binds to fibrillar and monomeric collagen in vitro. After the protein was labeled with near-infrared fluorescent dye, we injected the compound into mice fed on a NASH diet. Compared with that in control mice, the protein in these mice clears from the serum faster and accumulates significantly more in fibrotic livers. This work advances the development of targeted protein probes for in vivo fibrosis imaging.

Structural basis for the full and partial agonist activities of retinoid X receptor α ligands with an iso-butoxy and an isopropyl group.

Retinoid X receptors (RXRs) belong to a retinoid-binding subgroup of the nuclear receptor family, and their synthetic agonists have been developed as therapeutics for glucose and lipid metabolism, inflammation, and inflammatory bowel disease, although RXR agonists could cause side effects such as hypothyroidism, hypertriglyceridemia, and hepatomegaly. We previously reported novel full and partial agonists, NEt-3IB and NEt-4IB, which reduce the side effects, but the molecular basis of their different activity was not clear. In this study, we report the crystal structures of the ligand-binding domain of human RXRα complexed with NEt-3IB and NEt-4IB. Detailed comparisons of the two structures showed that the full agonist, NEt-3IB, is more stably accommodated in the ligand-binding pocket due to the interactions of the bulky iso-butoxy group with helices 5 and 7. The stabilization of these helices led to the stabilization of helix 12, which is important for formation of the coactivator-binding site. The structures shed light on the novel mechanism of the regulation of RXR activity through the interaction between the bound agonist and helix 7, an interaction that was not previously considered important.

The Increase in the Peroxidase Activity of the Cytochrome C with Substitutions in the Universal Binding Site Is Associated with Changes in the Ability to Interact with External Ligands.

Cytochrome c (CytC), a one-electron carrier, transfers electrons from complex bc1 to cytochrome c oxidase (CcO) in the electron-transport chain. Electrostatic interaction with the partners, complex bc1 and CcO, is ensured by a lysine cluster near the heme forming the Universal Binding Site (UBS). We constructed three mutant variants of mitochondrial CytC with one (2Mut), four (5Mut), and five (8Mut) Lys->Glu substitutions in the UBS and some compensating Glu->Lys substitutions at the periphery of the UBS for charge compensation. All mutants showed a 4-6 times increased peroxidase activity and accelerated binding of cyanide to the ferric heme of CytC. In contrast, decomposition of the cyanide complex with ferrous CytC, as monitored by magnetic circular dichroism spectroscopy, was slower in mutants compared to WT. Molecular dynamic simulations revealed the increase in the fluctuations of Cα atoms of individual residues of mutant CytC compared to WT, especially in the Ω-loop (70-85), which can cause destabilization of the Fe…S(Met80) coordination link, facilitation of the binding of exogenous ligands cyanide and peroxide, and an increase in peroxidase activity. It was found that only one substitution K72E is enough to induce all these changes, indicating the significance of K72 and the Ω-loop (70-85) for the structure and physiology of mitochondrial CytC. In this work, we also propose using a ferro-ferricyanide buffer as a substrate to monitor the peroxidase activity of CytC. This new approach allows us to determine the rate of peroxidase activity at moderate (200 µM) concentrations of H2O2 and avoid complications of radical formation during the reaction.

Binding characteristics and structural dynamics of two general odorant-binding proteins with plant volatiles in the olfactory recognition of Glyphodes pyloalis.

Glyphodes pyloalis Walker (Lepidoptera: Pyralidae) is the most destructive pest, causing severe damage to mulberry production in China's sericulture industry. The insecticide application in mulberry orchards poses a significant risk of poisoning to Bombyx mori. Shifting from insecticides to odor attractants is a beneficial alternative, but not much data is available on the olfactory system of G. pyloalis. We identified 114 chemosensory genes from the antennal transcriptome database of G. pyloalis, with 18 odorant-binding protein (OBP) and 17 chemosensory protein (CSP) genes significantly expressed in the antennae. Ligand-binding assays for two antennae-biased expressed general odorant-binding proteins (GOBPs) showed high binding affinities of GOBP1 to hexadecanal, ß-ionone, and 2-ethylhexyl acrylate, while GOBP2 exhibited binding to 4-tert-octylphenol, benzyl benzoate, ß-ionone, and farnesol. Computational simulations indicated that van der Waal forces predominantly contributed to the binding free energy in the binding processes of complexes. Among them, Phe12 of GOBP1 and Phe19 of GOBP2 were demonstrated to play crucial roles in their bindings to plant volatiles using site-directed mutagenesis experiments. Moreover, hexadecanal and ß-ionone attracted G. pyloalis male moths in the behavioral assays, while none of the candidate plant volatiles significantly affected female moths. Our findings provide a comprehensive understanding of the molecular mechanisms underlying olfactory recognition in G. pyloalis, setting the groundwork for novel mulberry pests control strategies based on insect olfaction.

Comprehensive characterization of a novel small-molecule activator for the nuclear receptor Nur77: Chemical, molecular, and biological insights.

The nuclear receptor Nur77 is a transcription factor belonging to the NR4A subfamily. Upon activation, it regulates a wide array of biological and pathophysiological processes by modulating the expression of its target genes. Previous findings have classified Nur77 as an orphan receptor because of the discovery of a structurally atypical ligand-binding domain and the lack of identification of an endogenous ligand. Nevertheless, recent studies have uncovered several endogenous, natural, and small synthetic molecules that can bind to and activate Nur77. However, developing selective and potent Nur77 activators remains a significant challenge. The discovery of novel and potential small synthetic molecules that modulate Nur77 activity will facilitate therapeutic interventions of Nur77 against several human diseases. In this study, we have reported the development of a novel and effective Nur77 ligand. Our data show that (1E,4E)-1,5-di(pyrazin-2-yl)penta-1,4-dien-3-one (PB) induces Nur77 transcriptional activity by interacting with a putative Nur77 ligand binding site by forming solid hydrogen bonding. Calculated chemical parameters denote that PB has sophisticated chemical features that will enhance its interaction with the Nur77 ligand-binding domain. Molecular docking simulations showed that PB fits in the Nur77 putative ligand binding site with solid hydrogen bonding, and molecular dynamics simulations indicate that PB binding would stabilize the Nur77 ligand binding domain. Further, in vitro studies revealed that PB increases Nur77 nuclear expression and activity, inhibits cigarette smoke-induced inflammatory phenotype of airway epithelial cells, and protects against apoptosis. These findings provide insights into developing an effective Nur77 small-molecule activator which could be developed into a therapeutic agent against inflammatory diseases.

Subtype-Specific Ligand Binding and Activation Gating in Homomeric and Heteromeric P2X Receptors.

P2X receptors are ATP-activated, non-specific cation channels involved in sensory signalling, inflammation, and certain forms of pain. Investigations of agonist binding and activation are essential for comprehending the fundamental mechanisms of receptor function. This encompasses the ligand recognition by the receptor, conformational changes following binding, and subsequent cellular signalling. The ATP-induced activation of P2X receptors is further influenced by the concentration of Mg2+ that forms a complex with ATP. To explore these intricate mechanisms, two new fluorescently labelled ATP derivatives have become commercially available: 2-[DY-547P1]-AHT-ATP (fATP) and 2-[DY-547P1]-AHT-α,ßMe-ATP (α,ßMe-fATP). We demonstrate a subtype-specific pattern of ligand potency and efficacy on human P2X2, P2X3, and P2X2/3 receptors with distinct relations between binding and gaiting. Given the high in vivo concentrations of Mg2+, the complex formed by Mg2+ and ATP emerges as an adequate ligand for P2X receptors. Utilising fluorescent ligands, we observed a Mg2+-dependent reduction in P2X2 receptor activation, while binding remained surprisingly robust. In contrast, P2X3 receptors initially exhibited decreased activation at high Mg2+ concentrations, concomitant with increased binding, while the P2X2/3 heteromer showed a hybrid effect. Hence, our new fluorescent ATP derivatives are powerful tools for further unravelling the mechanism underlying ligand binding and activation gating in P2X receptors.

Binding characterization of odorant-binding protein BhorOBP29 in Batocera horsfieldi (Hope) with host-plant volatiles.

Odorant binding proteins (OBPs) are involved in odorant discrimination and act as the first filter in the peripheral olfactory system. Previous studies have shown that BhorOBP29 is potentially involved in olfactory perception in an important wood-boring pest Batocera horsfieldi (Hope) (Coleoptera: Cerambycidae), however, its function remains unclear. Here, we investigated the ligand-binding profiles of recombinant BhorOBP29 with 22 compounds from its host plant using fluorescence competitive binding assays and fluorescence quenching assays. The results showed that BhorOBP29 could bind to five ligands relying mainly on hydrophobic interactions. Molecular docking analysis indicated that residues Ile48, Leu51, Met52, Trp57, Asn105, and Val119 were extensively involved in the interactions between BhorOBP29 and the five ligands. Furthermore, the site-directed mutagenesis analysis revealed that Leu51 and Met52 residues were indispensable for BhorOBP29-ligands binding. Finally, electroantennogram (EAG) assays confirmed that hexanal, (-)-limonene, and 2-methylbutyraldehyde elicited a concentration-dependent EAG response with a maximum at the concentration of 1/10 v/v. These findings suggest that BhorOBP29 may play a significant role in the perception of host plant volatiles by B. horsfieldi. This study may help to discover novel behavioral regulation and environmentally friendly strategies for controlling B. horsfieldi in the future.

[Advances in using artificial intelligence for predicting protein-ligand binding affinity].

The binding of proteins and ligands is a crucial aspect of life processes. The calculation of the protein-ligand binding affinity (PLBA) offers valuable insights into protein function, drug screening targets protein receptors, and enzyme modifications. In recent years, artificial intelligence (AI) has experienced rapid advancements, becoming widely used in PLBA prediction. This is attributed to its robust feature extraction ability, superior algorithm accuracy, and speedy calculations. Our paper aims to provide a comprehensive overview of AI predication process, associated resources, application scenarios, challenges, and potential solutions, serving as a valuable reference for the relevant research endeavors.

A novel and selective fluorescent ligand for the study of adenosine A2B receptors.

Fluorescent ligands have proved to be powerful tools in the study of G protein-coupled receptors in living cells. Here we have characterized a new fluorescent ligand PSB603-BY630 that has high selectivity for the human adenosine A2B receptor (A2BR). The A2BR appears to play an important role in regulating immune responses in the tumor microenvironment. Here we have used PSB603-BY630 to monitor specific binding to A2BRs in M1- and M2-like macrophages derived from CD14+ human monocytes. PSB603-BY630 bound with high affinity (18.3 nM) to nanoluciferase-tagged A2BRs stably expressed in HEK293G cells. The ligand exhibited very high selectivity for the A2BR with negligible specific-binding detected at NLuc-A2AR, NLuc-A1R, or NLuc-A3R receptors at concentrations up to 500 nM. Competition binding studies showed the expected pharmacology at A2BR with the A2BR-selective ligands PSB603 and MRS-1706 demonstrating potent inhibition of the specific binding of 50 nM PSB603-BY630 to A2BR. Functional studies in HEK293G cells using Glosensor to monitor Gs-coupled cyclic AMP responses indicated that PSB603-BY630 acted as a negative allosteric regular of the agonist responses to BAY 60-6583. Furthermore, flow cytometry analysis confirmed that PSB603-BY630 could be used to selectively label endogenous A2BRs expressed on human macrophages. This ligand should be an important addition to the library of fluorescent ligands which are selective for the different adenosine receptor subtypes, and will enable study of the role of A2BRs on immune cells in the tumor microenvironment.

A review of biophysical strategies to investigate protein-ligand binding: What have we employed?

The protein-ligand binding frequently occurs in living organisms and plays a crucial role in the execution of the functions of proteins and drugs. It is also an indispensable part of drug discovery and screening. While the methods for investigating protein-ligand binding are diverse, each has its own objectives, strengths, and limitations, which all influence the choice of method. Many studies concentrate on one or a few specific methods, suggesting that comprehensive summaries are lacking. Therefore in this review, these methods are comprehensively summarized and are discussed in detail: prediction and simulation methods, thermal and thermodynamic methods, spectroscopic methods, methods of determining three-dimensional structures of the complex, mass spectrometry-based methods and others. It is also important to integrate these methods based on the specific objectives of the research. With the aim of advancing pharmaceutical research, this review seeks to deepen the understanding of the protein-ligand binding process.

The affinity-efficacy problem: an essential part of pharmacology education.

A fundamental mistake in receptor theory has led to an enduring misunderstanding of how to estimate the affinity and efficacy of an agonist. These properties are inextricably linked and cannot be easily separated in any case where the binding of a ligand induces a conformation change in its receptor. Consequently, binding curves and concentration-response relationships for receptor agonists have no straightforward interpretation. This problem-the affinity-efficacy problem-remains overlooked and misunderstood despite it being recognized in 1987. To avoid the further propagation of this misunderstanding, we propose in this review that the affinity-efficacy problem should be included in the core curricula for pharmacology undergraduates proposed by the British Pharmacological Society and the International Union of Basic and Clinical Pharmacology (IUPHAR).

A Data Driven Strategy and Case Study for Implementation of Singlicate Analysis in Ligand Binding Assays Used for PK Quantitation.

Duplicate analysis has been a conventional practice in the industry for ligand-binding assays (LBA), particularly for plate-based platforms like Enzyme-linked immunosorbent assay (ELISA) and Meso Scale Discovery (MSD) assays. Recent whitepapers and guidance have opened a door to exploring the implementation of single-well (singlicate) analysis approach for LBAs. Although the bioanalytical industry has actively investigated the suitability of singlicate analysis, applications in supporting regulated LBA bioanalysis are limited. The primary reason for this limitation is the absence of appropriate strategy to facilitate the transition from duplicate to singlicate analysis. In this paper we present the first case study with our data-driven approach to implement singlicate analysis in a clinical pharmacokinetics (PK) plate based LBA assay with ISR data. The central aspect of this strategy is a head-to-head comparison with Precision and Accuracy assessment in both duplicate and singlicate formats as the initial stage of assay validation. Subsequently, statistical analysis is conducted to evaluate method variability in both precision and accuracy. The results of our study indicated that there was no impactful difference between duplicate vs singlicate, affirming the suitability of singlicate analysis for the remaining steps of PK assay validation. The validation results obtained through singlicate analysis demonstrated acceptable assay performance characteristics across all validation parameters, aligning with regulatory guidance. The validated PK assay in singlicate has been employed to support a Phase I study. The appropriateness of singlicate analyses is further supported by initial Incurred Sample Reanalysis (ISR) data in which 90.1% of ISR samples fall within the acceptable criteria.

Signal integration and adaptive sensory diversity tuning in Escherichia coli chemotaxis.

In uncertain environments, phenotypic diversity can be advantageous for survival. However, as the environmental uncertainty decreases, the relative advantage of having diverse phenotypes decreases. Here, we show how populations of E. coli integrate multiple chemical signals to adjust sensory diversity in response to changes in the prevalence of each ligand in the environment. Measuring kinase activity in single cells, we quantified the sensitivity distribution to various chemoattractants in different mixtures of background stimuli. We found that when ligands bind uncompetitively, the population tunes sensory diversity to each signal independently, decreasing diversity when the signal's ambient concentration increases. However, among competitive ligands, the population can only decrease sensory diversity one ligand at a time. Mathematical modeling suggests that sensory diversity tuning benefits E. coli populations by modulating how many cells are committed to tracking each signal proportionally as their prevalence changes.

Cryo2RT: a high-throughput method for room-temperature macromolecular crystallography from cryo-cooled crystals.

Advances in structural biology have relied heavily on synchrotron cryo-crystallography and cryogenic electron microscopy to elucidate biological processes and for drug discovery. However, disparities between cryogenic and room-temperature (RT) crystal structures pose challenges. Here, Cryo2RT, a high-throughput RT data-collection method from cryo-cooled crystals that leverages the cryo-crystallography workflow, is introduced. Tested on endothiapepsin crystals with four soaked fragments, thaumatin and SARS-CoV-2 3CLpro, Cryo2RT reveals unique ligand-binding poses, offers a comparable throughput to cryo-crystallography and eases the exploration of structural dynamics at various temperatures.

Identification and Functional Analysis of Odorant Binding Proteins in Apriona germari (Hope).

Apriona germari (Hope) presents a significant threat as a dangerous wood-boring pest, inflicting substantial harm to forest trees. Investigating the olfactory sensory system of A. germari holds substantial theoretical promise for developing eco-friendly control strategies. To date, however, the olfactory perception mechanism in A. germari remains largely unknown. Therefore, we performed transcriptome sequencing of A. germari across four distinct body parts: antennae, foreleg tarsal segments, mouthparts (maxillary and labial palps), and abdomen terminals, pinpointing the odorant binding protein (OBP) genes and analyzing their expression. We found eight AgerOBPs (5, 19, 23, 25, 29, 59, 63, 70) highly expressed in the antennae. In our competitive binding experiments, AgerOBP23 showed strong binding abilities to the pheromone component fuscumol acetate, eight plant volatiles (farnesol, cis-3-hexenal, nerolidol, myristol acetate, cis-3-hexenyl benzoate, (-)-α-cedrene, 3-ethylacetophenone, and decane), and four insecticides (chlorpyrifos, phoxim, indoxacarb, and cypermethrin). However, AgerOBP29 and AgerOBP63 did not show prominent binding activities to these tested chemicals. Through homology modeling and molecular docking, we identified the key amino acid sites involved in the binding process of AgerOBP23 to these ligands, which shed light on the molecular interactions underlying its binding specificity. Our study suggests that AgerOBP23 may serve as a potential target for future investigations of AgerOBP ligand binding. This approach is consistent with the reverse chemical ecology principle, establishing the groundwork for future studies focusing on attractant or repellent development by exploring further the molecular interactions between OBP and various compounds.

Female-Biased Odorant Receptor MmedOR48 in the Parasitoid Microplitis mediator Broadly Tunes to Plant Volatiles.

Odorant receptors (ORs) play a crucial role in insect chemoreception. Here, a female-biased odorant receptor MmedOR48 in parasitoid Microplitis mediator was fully functionally characterized. The qPCR analysis suggested that the expression level of MmedOR48 increased significantly after adult emergence and was expressed much more in the antennae. Moreover, an in situ hybridization assay showed MmedOR48 was extensively located in the olfactory sensory neurons. In two-electrode voltage clamp recordings, recombinant MmedOR48 was broadly tuned to 23 kinds of volatiles, among which five plant aldehyde volatiles excited the strongest current recording values. Subsequent molecular docking analysis coupled with site-directed mutagenesis demonstrated that key amino acid residues Thr142, Gln80, Gln282, and Thr312 together formed the binding site in the active pocket for the typical aldehyde ligands. Furthermore, ligands of MmedOR48 could stimulate electrophysiological activities in female adults of the M. mediator. The main aldehyde ligand, nonanal, aroused significant behavioral preference of M. mediator in females than in males. These findings suggest that MmedOR48 may be involved in the recognition of plant volatiles in M. mediator, which provides valuable insight into understanding the olfactory mechanisms of parasitoids.

Identification of small-molecule ligand-binding sites on and in the ARNT PAS-B domain.

Transcription factors are challenging to target with small-molecule inhibitors due to their structural plasticity and lack of catalytic sites. Notable exceptions include naturally ligand-regulated transcription factors, including our prior work with the hypoxia-inducible factor (HIF)-2 transcription factor, showing that small-molecule binding within an internal pocket of the HIF-2α Per-Aryl hydrocarbon Receptor Nuclear Translocator (ARNT)-Sim (PAS)-B domain can disrupt its interactions with its dimerization partner, ARNT. Here, we explore the feasibility of targeting small molecules to the analogous ARNT PAS-B domain itself, potentially opening a promising route to modulate several ARNT-mediated signaling pathways. Using solution NMR fragment screening, we previously identified several compounds that bind ARNT PAS-B and, in certain cases, antagonize ARNT association with the transforming acidic coiled-coil containing protein 3 transcriptional coactivator. However, these ligands have only modest binding affinities, complicating characterization of their binding sites. We address this challenge by combining NMR, molecular dynamics simulations, and ensemble docking to identify ligand-binding "hotspots" on and within the ARNT PAS-B domain. Our data indicate that the two ARNT/transforming acidic coiled-coil containing protein 3 inhibitors, KG-548 and KG-655, bind to a ß-sheet surface implicated in both HIF-2 dimerization and coactivator recruitment. Furthermore, while KG-548 binds exclusively to the ß-sheet surface, KG-655 can additionally bind within a water-accessible internal cavity in ARNT PAS-B. Finally, KG-279, while not a coactivator inhibitor, exemplifies ligands that preferentially bind only to the internal cavity. All three ligands promoted ARNT PAS-B homodimerization, albeit to varying degrees. Taken together, our findings provide a comprehensive overview of ARNT PAS-B ligand-binding sites and may guide the development of more potent coactivator inhibitors for cellular and functional studies.

Structural and functional characterization of the IpaD π-helix reveals critical roles in DOC interaction, T3SS apparatus maturation, and Shigella virulence.

Shigella spp. are highly pathogenic members of the Enterobacteriaceae family, causing ∼269 million cases of bacillary dysentery and >200,000 deaths each year. Like many Gram-negative pathogens, Shigella rely on their type three secretion system (T3SS) to inject effector proteins into eukaryotic host cells, driving both cellular invasion and evasion of host immune responses. Exposure to the bile salt deoxycholate (DOC) significantly enhances Shigella virulence and is proposed to serve as a critical environmental signal present in the small intestine that prepares Shigella's T3SS for efficient infection of the colonic epithelium. Here, we uncover critical mechanistic details of the Shigella-specific DOC signaling process by describing the role of a π-helix secondary structure element within the T3SS tip protein invasion plasmid antigen D (IpaD). Biophysical characterization and high-resolution structures of IpaD mutants lacking the π-helix show that it is not required for global protein structure, but that it defines the native DOC binding site and prevents off target interactions. Additionally, Shigella strains expressing the π-helix deletion mutants illustrate the pathogenic importance of its role in guiding DOC interaction as flow cytometry and gentamycin protection assays show that the IpaD π-helix is essential for DOC-mediated apparatus maturation and enhanced invasion of eukaryotic cells. Together, these findings add to our understanding of the complex Shigella pathogenesis pathway and its evolution to respond to environmental bile salts by identifying the π-helix in IpaD as a critical structural element required for translating DOC exposure to virulence enhancement.

Bivalent affinity binding-inspired PPARγ immobilization with selective conformation and improved ligand-binding activity.

Functional protein immobilization forms the basis for bio-detections. A series of one-point, site-specific immobilization methods have been developed, however, it still remains as a challenge how to avoid the proteins to move in all directions as well as conveniently regenerate the bio-devices. Herein, we have developed a bivalent affinity binding-inspired method for PPARγ immobilization using DNA aptamer and nickel-nitrilotriacetic acid (Ni2+-NTA) chelation. The specific DNA aptamer (Apt 2) was selected by an on-column systematic evolution of ligands by exponential enrichment (SELEX) method with affinity of (1.57 ± 0.15) × 105 M-1, determined by isothermal titration calorimetry (ITC). Apt 2 and nickel-nitrilotriacetic acid (Ni2+-NTA) were modified on macroporous silica gels via L-α-allylglycine as a linker. They respectively interacted with PPARγ and 6×His tag via bivalent affinity binding for the receptor immobilization. After comprehensive surface characterization, PPARγ was proved to be successful immobilized. Chromatographic studies revealed that the immobilized PPARγ has conformation selectivity, which discriminated agonist and antagonist of the receptor. Ligand-binding parameters (affinity and rate constant) of four agonists (rosiglitazone, pioglitazone, troglitazone, and magnolol) with PPARγ were determined. Troglitazone showed the lowest dissociation rate constant. The binding affinities (3.28 × 107, 1.91 × 106, 2.25 × 107, and 2.43 × 107 M-1) were highly consistent with the data obtained using purified receptor in solution (2.16 × 107, 4.52 × 106, 1.20 × 107, and 1.56 × 107 M-1), offering reliable bio-detection method for PPARγ and its ligands. Due to the biocompatibility of nuclear receptor with DNA, it is conceivable that the bivalent affinity-based method will be a general method for the immobilization of other nuclear receptors, which may provide selective conformation and improved ligand-binding activity for the receptors.