In Vitro Characterization of an O-Specific Glycosyltransferase Involved in Flagellin Glycosylation.

Glycosyltransferases play a fundamental role in the biosynthesis of glycoproteins and glycotherapeutics. In this study, we investigated protein glycosyltransferase FlgGT1, belonging to the GT2 family. The GT2 family includes cysteine S-glycosyltransferases involved in antimicrobial peptide biosyntheses, sharing conserved catalytic domains while exhibiting diverse C-terminal domains. Our in vitro studies revealed that FlgGT1 recognizes structural motifs rather than specific amino acid sequences when glycosylating the flagellin protein Hag. Notably, FlgGT1 is selective for serine or threonine O-glycosylation over cysteine S-glycosylation. Molecular dynamics simulations provided insights into the structural basis of FlgGT1's ability to accommodate various sugar nucleotides as donor substrates. Mutagenesis experiments on FlgGT1 demonstrated that truncating the relatively large C-terminal domain resulted in a loss of flagellin glycosylation activity. Our classification based on sequence similarity network analysis and AlphaFold2 structural predictions suggests that the acquisition of the C-terminal domain is a key evolutionary adaptation conferring distinct substrate specificities on glycosyltransferases within the GT2 family.

Comprehensive Epitope Analysis of Monoclonal Antibodies Binding to Hen Egg Ovalbumin Using a Peptide Array.

Food allergies are a significant health issue worldwide. In many countries, labeling of primary allergens in food products has been made mandatory to ensure consumer safety. In food manufacturing settings, the lateral flow immunoassay (LFI)-based on antigen-antibody reactions-is a rapid and accurate method for allergen testing and is widely used. Peptide arrays are tools that enable the synthesis of peptides of any sequence on a substrate and high-throughput analysis of their interactions with chemicals. This study aimed to investigate a new application of peptide arrays in the field of food technology, particularly in the development of antibodies for food allergen testing. First, monoclonal antibodies against hen egg ovalbumin, a major food allergen, were produced. Then, using a peptide array, the epitope and specificity of the antibodies were comprehensively and precisely analyzed. Finally, an LFI kit incorporating the antibodies demonstrated both high specificity and detection sensitivity for food allergen testing. These findings indicate that peptide arrays are valuable tools in the development of antibodies for food allergen testing, ensuring reliability and accuracy at the molecular level.

Structure Prediction and Genome Mining-Aided Discovery of the Bacterial C-Terminal Tryptophan Prenyltransferase PalQ.

Post-translational prenylations, found in eukaryotic primary metabolites and bacterial secondary metabolites, play crucial roles in biomolecular interactions. Employing genome mining methods combined with AlphaFold2-based predictions of protein interactions, PalQ , a prenyltransferase responsible for the tryptophan prenylation of RiPPs produced by Paenibacillus alvei, is identified. PalQ differs from cyanobactin prenyltransferases because of its evolutionary relationship to isoprene synthases, which enables PalQ to transfer extended prenyl chains to the indole C3 position. This prenylation introduces structural diversity to the tryptophan side chain and also leads to conformational dynamics in the peptide backbone, attributed to the cis/trans isomerization that arises from the formation of a pyrrolidine ring. Additionally, PalQ exhibited pronounced positional selectivity for the C-terminal tryptophan. Such enzymatic characteristics offer a toolkit for peptide therapeutic lipidation.

Association of NOTCH3 With Elastic Fiber Dispersion in the Infrarenal Abdominal Aorta of Cynomolgus Monkeys.

BACKGROUND: The regional heterogeneity of vascular components and transcriptomes is an important determinant of aortic biology. This notion has been explored in multiple mouse studies. In the present study, we examined the regional heterogeneity of aortas in nonhuman primates. METHODS: Aortic samples were harvested from the ascending, descending thoracic, suprarenal, and infrarenal regions of young control monkeys and adult monkeys with high fructose consumption for 3 years. The regional heterogeneity of aortic structure and transcriptomes was examined by histological and bulk RNA sequencing analyses, respectively. RESULTS: Immunostaining of CD31 and αSMA (alpha-smooth muscle actin) revealed that endothelial and smooth muscle cells were distributed homogeneously across the aortic regions. In contrast, elastic fibers were less abundant and dispersed in the infrarenal aorta compared with other regions and associated with collagen deposition. Bulk RNA sequencing identified a distinct transcriptome related to the Notch signaling pathway in the infrarenal aorta with significantly increased NOTCH3 mRNA compared with other regions. Immunostaining revealed that NOTCH3 protein was increased in the media of the infrarenal aorta. The abundance of medial NOTCH3 was positively correlated with the dispersion of elastic fibers. Adult cynomolgus monkeys with high fructose consumption displayed vascular wall remodeling, such as smooth muscle cell loss and elastic fiber disruption, predominantly in the infrarenal region. The correlation between NOTCH3 and elastic fiber dispersion was enhanced in these monkeys. CONCLUSIONS: Aortas of young cynomolgus monkeys display regional heterogeneity of their transcriptome and the structure of elastin and collagens. Elastic fibers in the infrarenal aorta are dispersed along with upregulation of medial NOTCH3.

Structural and functional analysis of hyper-thermostable ancestral L-amino acid oxidase that can convert Trp derivatives to D-forms by chemoenzymatic reaction.

Production of D-amino acids (D-AAs) on a large-scale enables to provide precursors of peptide therapeutics. In this study, we designed a novel L-amino acid oxidase, HTAncLAAO2, by ancestral sequence reconstruction, exhibiting high thermostability and long-term stability. The crystal structure of HTAncLAAO2 was determined at 2.2 Å by X-ray crystallography, revealing that the enzyme has an octameric form like a "ninja-star" feature. Enzymatic property analysis demonstrated that HTAncLAAO2 exhibits three-order larger kcat/Km values towards four L-AAs (L-Phe, L-Leu, L-Met, and L-Ile) than that of L-Trp. Through screening the variants, we obtained the HTAncLAAO2(W220A) variant, which shows a > 6-fold increase in kcat value toward L-Trp compared to the original enzyme. This variant applies to synthesizing enantio-pure D-Trp derivatives from L- or rac-forms at a preparative scale. Given its excellent properties, HTAncLAAO2 would be a starting point for designing novel oxidases with high activity toward various amines and AAs.

Association of NOTCH3 with Elastic Fiber Dispersion in the Infrarenal Abdominal Aorta of Cynomolgus Monkeys.

Background: The regional heterogeneity of vascular components and transcriptomes is an important determinant of aortic biology. This notion has been explored in multiple mouse studies. In the present study, we examined the regional heterogeneity of aortas in non-human primates. Methods: Aortic samples were harvested from the ascending, descending, suprarenal, and infrarenal regions of young control monkeys and adult monkeys provided with high fructose for 3 years. The regional heterogeneity of aortic structure and transcriptomes was examined by histological and bulk RNA sequencing analyses. Results: Immunostaining of CD31 and αSMA revealed that endothelial and smooth muscle cells were distributed homogeneously across the aortic regions. In contrast, elastic fibers were less abundant and dispersed in the infrarenal aorta compared to other regions and associated with collagen deposition. Bulk RNA sequencing identified a distinct transcriptome related to the Notch signaling pathway in the infrarenal aorta with significantly increased NOTCH3 mRNA compared to other regions. Immunostaining revealed that NOTCH3 protein was increased in the media of the infrarenal aorta. The abundance of medial NOTCH3 was positively correlated with the dispersion of elastic fibers. Adult cynomolgus monkeys provided with high fructose displayed vascular wall remodeling, such as smooth muscle cell loss and elastic fiber disruption, predominantly in the infrarenal region. The correlation between NOTCH3 and elastic fiber dispersion was enhanced in these monkeys. Conclusions: Aortas of young cynomolgus monkeys display regional heterogeneity of their transcriptome and the structure of elastin and collagens. Elastic fibers in the infrarenal aorta are dispersed along with upregulation of medial NOTCH3. HIGHLIGHTS: - The present study determined the regional heterogeneity of aortas from cynomolgus monkeys.- Aortas of young cynomolgus monkeys displayed region-specific aortic structure and transcriptomes.- Elastic fibers were dispersed in the infrarenal aorta along with increased NOTCH3 abundance in the media.

Differential prediction of high-sensitivity cardiac troponin-I, but not N-terminal pro-brain natriuretic peptide, in different pitavastatin doses on cardiovascular events in stable coronary artery disease.

BACKGROUND: This study aimed to examine whether high-sensitivity cardiac troponin-I (hsTnI) and N-terminal pro-brain natriuretic peptide (NT-proBNP) could predict future major adverse cardiovascular events (MACE) in stable coronary artery disease (CAD) patients with high- or low-dose of pitavastatin. METHODS: This was a case-cohort analysis of the REAL-CAD study, a randomized trial of high- or low-dose (4 or 1 mg/day) pitavastatin therapy in patients with stable CAD. We examined the MACE risk according to the quartile of hsTnI and NT-proBNP at baseline. RESULTS: A total of 1336 and 1396 patients including 582 MACE cases were randomly examined into the hsTnI and NT-proBNP cohort, respectively. Both higher levels of hsTnI and NT-proBNP at baseline were significantly associated with increased risk of MACE (p < 0.001, respectively). When separately analyzed in statin dose, the higher marker levels were significantly associated with higher MACE risk in all cohorts (p < 0.001 in all cohorts). After multivariable adjustment, hsTnI levels were significantly associated with MACE risk in low-dose statin group (HR 2.54, p = 0.0001); however, in high-dose pitavastatin therapy, a significant association was diminished in MACE risk among the quartiles of baseline hsTnI levels (p = 0.154). Conversely in the NT-proBNP cohort, the association between NT-proBNP levels and MACE risk was constantly observed regardless of pitavastatin dose even after multivariable adjustment (both p < 0.0001). CONCLUSIONS: Patients with high hsTnI levels had high risk of MACE in low-dose statin group, but not in high-dose, suggesting that high-dose statin treatment might decrease MACE risk in stable CAD patients with high hsTnI levels.

Functional Exploration of Conserved Sequences in the Distal Face of Angiotensinogen-Brief Report.

BACKGROUND: Angiotensinogen (AGT) is an essential component in the renin-angiotensin system. AGT has highly conserved sequences in the loop and ß-sheet regions among species; however, their functions have not been studied. METHODS: Adeno-associated viral vector (AAV) serotype 2/8 encoding mouse AGT with mutations of conserved sequences in the loop (AAV.loop-Mut), ß-sheet (AAV.ßsheet-Mut), or both regions (AAV.loop/ßsheet-Mut) was injected into male hepatocyte-specific AGT-deficient (hepAGT-/-) mice in an LDL (low-density lipoprotein) receptor-deficient background. AAV containing mouse wild-type AGT (AAV.mAGT) or a null vector (AAV.null) were used as controls. Two weeks after AAV administration, all mice were fed a western diet for 12 weeks. To determine how AGT secretion is regulated in hepatocytes, AAVs containing the above mutations were transducted into HepG2 cells. RESULTS: In hepAGT-/- mice infected with AAV.loop-Mut or ßsheet-Mut, plasma AGT concentrations, systolic blood pressure, and atherosclerosis were comparable to those in AAV.mAGT-infected mice. Interestingly, plasma AGT concentrations, systolic blood pressure, and atherosclerotic lesion size in hepAGT-/- mice infected with AAV.loop/ßsheet-Mut were not different from mice infected with AAV.null. In contrast, hepatic Agt mRNA abundance was elevated to a comparable magnitude as AAV.mAGT-infected mice. Immunostaining showed that AGT protein was accumulated in hepatocytes of mice infected with AAV.loop/ßsheet-Mut or HepG2 cells transducted with AAV.loop/ßsheet-Mut. Accumulated AGT was not located in the endoplasmic reticulum. CONCLUSIONS: The conserved sequences in either the loop or ß-sheet region individually have no effect on AGT regulation, but the conserved sequences in both regions synergistically contribute to the secretion of AGT from hepatocytes.

Ligand Screening System for the RXRα Heterodimer Using the Fluorescence RXR Agonist CU-6PMN.

Retinoid X receptor (RXR), a nuclear receptor (NR) that regulates transcription of target genes in a ligand binding-dependent manner, is of interest as a drug target. RXR agonists have been developed as therapeutic agents for cutaneous invasive T-cell lymphoma (e.g., bexarotene (1)) and investigated as potential anti-inflammatory agents. Screening systems for the binding of RXR alone have been reported. However, although RXRs function as RXR heterodimers, information on systems to evaluate the differential binding of RXR agonists as RXR heterodimers has not been available until recently. Here we show that the fluorescent RXR agonist CU-6PMN (3), designed by our group, can be useful for assessing RXR binding to PPARγ/RXRα, and that the binding data differ from those of RXRα alone. This screening method opens a new avenue for binding assays for RXR heterodimers.

Definition of an Index Parameter To Screen Highly Functional Enzymes Derived from a Biochemical and Thermodynamic Analysis of Ancestral meso-Diaminopimelate Dehydrogenases.

Sequence-based protein design approaches are being adopted to generate highly functional enzymes; however, screening the enzymes remains a time-consuming task. In this study, by analyzing the enzymatic properties of four ancestral meso-2,6-diaminopimelate dehydrogenases (AncDAPDHs), AncDAPDH-N1, -N2, -N3, and -N4, we attempted to define a new index parameter that is helpful for efficiently screening the enzymes. Biochemical and thermodynamic analyses indicated that only AncDAPDH-N4 exhibited greater thermal stability than and activity similar to those of native DAPDHs. Structural and sequence comparisons between DAPDH from Corynebacterium glutamicum (CgDAPDH) and the AncDAPDHs suggested that "quality of mutations" is a potential index parameter. In fact, the mutations introduced from CgDAPDH to AncDAPDH-N4 correlated highly with the mutations accumulated during the evolution process from mesophiles to thermophiles. These results suggest that, although there are several exceptions, the correlation coefficient can be used as an index parameter for screening high-functioning enzymes from sequence data.

Reaction Mechanism of Ancestral l-Lys α-Oxidase from Caulobacter Species Studied by Biochemical, Structural, and Computational Analysis.

The flavin-dependent amine oxidase superfamily contains various l-amino acid oxidases (LAAOs) bearing different substrate specificities and enzymatic properties. LAAOs catalyze the oxidation of the α-amino group of l-amino acids (L-AAs) to produce imino acids and H2O2. In this study, an ancestral l-Lys α-oxidase (AncLLysO2) was designed utilizing genome-mined sequences from the Caulobacter species. The AncLLysO2 exhibited high specificity toward l-Lys; the k cat/K m values toward l-Lys were one and two orders larger than those of l-Arg and l-ornithine, respectively. Liquid chromatography-high resolution mass spectrometry analysis indicated that AncLLysO2 released imino acid immediately from the active site after completion of oxidation of the α-amino group. Crystal structures of the ligand-free, l-Lys- and l-Arg-bound forms of AncLLysO2 were determined at 1.4-1.6 Å resolution, indicating that the active site of AncLLysO2 kept an open state during the reaction and more likely to release products. The structures also indicated the substrate recognition mechanism of AncLLysO2; ε-amino, α-amino, and carboxyl groups of l-Lys formed interactions with Q357, A551, and R77, respectively. Biochemical and molecular dynamics simulation analysis of AncLLysO2 indicated that active site residues that indirectly interact with the substrate are also important to exhibit high activity; for example, the aromatic group of Y219 is important to ensure that the l-Lys substrate is placed in the correct position to allow the reaction to proceed efficiently. Taken together, we propose the reaction mechanism of AncLLysO2.

Enzymatic Conjugation of Modified RNA Fragments by Ancestral RNA Ligase AncT4_2.

Oligonucleotide therapeutics have great potential as a next-generation approach to treating intractable diseases. Large quantities of modified DNA/RNA containing xenobiotic nucleic acids (XNAs) must be synthesized before clinical application. In this study, the ancestral RNA ligase AncT4_2 was designed by ancestral sequence reconstruction (ASR) to perform the conjugation reaction of modified RNA fragments. AncT4_2 had superior properties to native RNA ligase 2 from T4 phage (T4Rnl2), including high productivity, a >2.5-fold-higher turnover number, and >10°C higher thermostability. One remarkable point is the broad substrate selectivity of AncT4_2; the activity of AncT4_2 toward 17 of the modified RNA fragments was higher than that of T4Rnl2. The activity was estimated by measuring the conjugation reaction of two RNA strands, 3'-OH (12 bp) and 5'-PO4 (12 bp), in which the terminal and penultimate positions of the 3'-OH fragment and the first and second positions of the 5'-PO4 fragment were substituted by 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl, and 2'-H, respectively. The enzymatic properties of AncT4_2 allowed the enzyme to conjugate large quantities of double-stranded RNA coding for patisiran (>400 µM level), which was formed by four RNA fragments containing 2'-OMe-substituted nucleic acids. Structural analysis of modeled AncT4_2 suggested that protein dynamics were changed by mutation to Gly or indel during ASR and that this may positively impact the conjugation of modified RNA fragments with the enzyme. AncT4_2 is expected to be a key biocatalyst in synthesizing RNA therapeutics by an enzymatic reaction. IMPORTANCE RNA therapeutics is one of the next-generation medicines for treating various diseases. Our designed ancestral RNA ligase AncT4_2 exhibited excellent enzymatic properties, such as high thermal stability, productivity, specific activity, and broad substrate selectivity compared to native enzymes. These advantages create the potential for AncT4_2 to be applied in conjugating the modified RNA fragments containing various xenobiotic nucleic acids. In addition, patisiran, a known polyneuropathy therapeutic, could be synthesized from four fragmented oligonucleotides at a preparative scale. Taken together, these findings indicate AncT4_2 could open the door to synthesizing RNA therapeutics by enzymatic reaction at large-scale production.

Embryonic Heterogeneity of Smooth Muscle Cells in the Complex Mechanisms of Thoracic Aortic Aneurysms.

Smooth muscle cells (SMCs) are the major cell type of the aortic wall and play a pivotal role in the pathophysiology of thoracic aortic aneurysms (TAAs). TAAs occur in a region-specific manner with the proximal region being a common location. In this region, SMCs are derived embryonically from either the cardiac neural crest or the second heart field. These cells of distinct origins reside in specific locations and exhibit different biological behaviors in the complex mechanism of TAAs. The purpose of this review is to enhance understanding of the embryonic heterogeneity of SMCs in the proximal thoracic aorta and their functions in TAAs.

ß-Aminopropionitrile-induced aortic aneurysm and dissection in mice.

The mechanistic basis for the formation of aortic aneurysms and dissection needs to be elucidated to facilitate the development of effective medications. ß-Aminopropionitrile administration in mice has been used frequently to study the pathologic features and mechanisms of aortic aneurysm and dissection. This mouse model mimics several facets of the pathology of human aortic aneurysms and dissection, although many variables exist in the experimental design and protocols that must be resolved to determine its application to the human disease. In the present brief review, we have introduced the development of this mouse model and provided insights into understanding its pathologic features.

Imaging Techniques for Aortic Aneurysms and Dissections in Mice: Comparisons of Ex Vivo, In Situ, and Ultrasound Approaches.

Aortic aneurysms and dissections are life-threatening conditions that have a high risk for lethal bleeding and organ malperfusion. Many studies have investigated the molecular basis of these diseases using mouse models. In mice, ex vivo, in situ, and ultrasound imaging are major approaches to evaluate aortic diameters, a common parameter to determine the severity of aortic aneurysms. However, accurate evaluations of aortic dimensions by these imaging approaches could be challenging due to pathological features of aortic aneurysms. Currently, there is no standardized mode to assess aortic dissections in mice. It is important to understand the characteristics of each approach for reliable evaluation of aortic dilatations. In this review, we summarize imaging techniques used for aortic visualization in recent mouse studies and discuss their pros and cons. We also provide suggestions to facilitate the visualization of mouse aortas.

Thermostability enhancement of l-glutamate oxidase from Streptomyces sp. NT1 by full consensus protein design.

Thermostable l-glutamate oxidases (LGOXs) are desirable for use in l-glutamate (L-Glu) assay kits, enzymatic synthesis of α-ketoglutarate and for biosensor development. However, protein engineering efforts to improve thermostability often lead to a decrease in enzymatic activity. In this report, we aimed to enhance the thermostability (melting temperature, Tm) of a mesophilic LGOX from Streptomyces sp. NT1 (LGOXNT1) without a reduction in activity by a sequence-based protein design approach, termed full consensus (Fc) protein design. Among the 690 amino acids of LGOXNT1, 104 amino acids were substituted by the Fc protein design. The mutant gene was artificially synthesized and expressed in Escherichia coli BL21(DE3) cells. The Tm of the purified, recombinant LGOX mutant (FcLGOX) was determined to be ∼72 °C, which is an increase on the Tm of 65 °C for LGOXNT1 and the highest among known LGOXs. Importantly, purified FcLGOX showed no loss of specific activity or substrate specificity after a 30-min incubation at 70 °C. Our findings provide a new approach to improve the thermostability of enzymes.

Design of a Full-Consensus Glutamate Decarboxylase and Its Application to GABA Biosynthesis.

Glutamate decarboxylase (GAD) catalyses the decarboxylation of L-glutamate to gamma-aminobutyric acid (GABA). Improvement of the enzymatic properties of GAD is important for the low-cost synthesis of GABA. In this study, utilizing sequences of enzymes homologous with GAD from lactic acid bacteria, highly mutated GADs were designed using sequence-based protein design methods. Two mutated GADs, FcGAD and AncGAD, generated by full-consensus design and ancestral sequence reconstruction, had more desirable properties than native GADs. With respect to thermal stability, the half-life of the designed GADs was about 10 °C higher than that of native GAD. The productivity of FcGAD was considerably higher than those of known GADs; more than 250 mg/L of purified enzyme could be produced in the E. coli expression system. In a production test using 26.4 g of l-glutamate and 3.0 g of resting cells, 17.2 g of GABA could be prepared within one hour, without purification, in a one-pot synthesis.

Catalytic mechanism of ancestral L-lysine oxidase assigned by sequence data mining.

A large number of protein sequences are registered in public databases such as PubMed. Functionally uncharacterized enzymes are included in these databases, some of which likely have potential for industrial applications. However, assignment of the enzymes remained difficult tasks for now. In this study, we assigned a total of 28 original sequences to uncharacterized enzymes in the FAD-dependent oxidase family expressed in some species of bacteria including Chryseobacterium, Flavobacterium, and Pedobactor. Progenitor sequence of the assigned 28 sequences was generated by ancestral sequence reconstruction, and the generated sequence exhibited L-lysine oxidase activity; thus, we named the enzyme AncLLysO. Crystal structures of ligand-free and ligand-bound forms of AncLLysO were determined, indicating that the enzyme recognizes L-Lys by hydrogen bond formation with R76 and E383. The binding of L-Lys to AncLLysO induced dynamic structural change at a plug loop formed by residues 251 to 254. Biochemical assays of AncLLysO variants revealed the functional importance of these substrate recognition residues and the plug loop. R76A and E383D variants were also observed to lose their activity, and the kcat/Km value of G251P and Y253A mutations were approximately 800- to 1800-fold lower than that of AncLLysO, despite the indirect interaction of the substrates with the mutated residues. Taken together, our data demonstrate that combinational approaches to sequence classification from database and ancestral sequence reconstruction may be effective not only to find new enzymes using databases of unknown sequences but also to elucidate their functions.

Partial Consensus Design and Enhancement of Protein Function by Secondary-Structure-Guided Consensus Mutations.

Consensus design (CD) is a representative sequence-based protein design method that enables the design of highly functional proteins by analyzing vast amounts of protein sequence data. This study proposes a partial consensus design (PCD) of a protein as a derivative approach of CD. The method replaces the target protein sequence with a consensus sequence in a secondary-structure-dependent manner (i.e., regionally dependent and divided into α-helix, ß-sheet, and loop regions). In this study, we generated several artificial partial consensus l-threonine 3-dehydrogenases (PcTDHs) by PCD using the TDH from Cupriavidus necator (CnTDH) as a target protein. Structural and functional analysis of PcTDHs suggested that thermostability would be independently improved when consensus mutations are introduced into the loop region of TDHs. On the other hand, enzyme kinetic parameters (kcat/Km) and average productivity would be synergistically enhanced by changing the combination of the mutations-replacement of one region of CnTDH with a consensus sequence provided only negative effects, but the negative effects were nullified when the two regions were replaced simultaneously. Taken together, we propose the hypothesis that there are protein regions that encode individual protein properties, such as thermostability and activity, and that the introduction of consensus mutations into these regions could additively or synergistically modify their functions.

Creation of Fluorescent RXR Antagonists Based on CBTF-EE and Application to a Fluorescence Polarization Binding Assay.

Retinoid X receptor (RXR) ligands often bind in modes in which the carboxy group forms a hydrogen bond inside the ligand-binding pocket (LBP). However, our previously reported RXR antagonist, CBTF-EE (4a), binds with its carboxy group directed outside the LBP and its alkoxy side chain located inside the LBP. Here, we examined the binding modes of 4b and 4c bearing a nitrobenzoxadiazole (NBD) or boron-dipyrromethene (BODIPY) fluorophore, respectively, at the end of the alkoxy chain of 4a. Both compounds function as RXR antagonists. 4c, but not 4b, was available for a fluorescence polarization binding assay, indicating that rotation of BODIPY, but not NBD, is restricted in the bound state. The fluorescence findings, supported by docking simulations, suggest the fluorophores are located outside the LBP, so that the binding mode of 4b and 4c is different from that of 4a. The assay results were highly correlated with those of a [3H]9-cis-retinoic acid assay.