Sosuke Yoshinaga

Hyrtioreticulins A-E, indole alkaloids inhibiting the ubiquitin-activating enzyme, from the marine sponge Hyrtios reticulatus.

Hyrtioreticulins A-E (1-5) were isolated from the marine sponge Hyrtios reticulatus, along with a known alkaloid, hyrtioerectine B (6). Structural elucidation on the basis of spectral data showed that 1, 2, and 5 are new tetrahydro-β-carboline alkaloids, while 3 and 4 are new azepinoindole-type alkaloids. Hyrtioreticulins A and B (1 and 2) inhibited ubiquitin-activating enzyme (E1) with IC(50) values of 0.75 and 11μg/mL, respectively, measured by their inhibitory abilities against the formation of an E1-ubiquitin intermediate. So far, only five E1 inhibitors, panapophenanthrine, himeic acid A, largazole, and hyrtioreticulins A and B (1 and 2), have been isolated from natural sources and, among them, 1 is the most potent E1 inhibitor.

Structure of the mouse sex peptide pheromone ESP1 reveals a molecular basis for specific binding to the class C G-protein-coupled vomeronasal receptor

Background: The male mouse-specific peptide pheromone ESP1 is recognized by a specific G-protein-coupled receptor (GPCR) and enhances female sexual receptive behavior. Results: The structural and functional domain of ESP1 was defined, and its structure and receptor-binding site were revealed. Conclusion: Electrostatic interactions determine the specific binding of ESP1 to its receptor. Significance: This study provides a basis for the narrowly tuned sensing of mammalian peptide pheromones. Exocrine gland-secreting peptide 1 (ESP1) is a sex pheromone that is released in male mouse tear fluids and enhances female sexual receptive behavior. ESP1 is selectively recognized by a specific class C G-protein-coupled receptor (GPCR), V2Rp5, among the hundreds of receptors expressed in vomeronasal sensory neurons (VSNs). The specific sensing mechanism of the mammalian peptide pheromone by the class C GPCR remains to be elucidated. Here we identified the minimal functional region needed to retain VSN-stimulating activity in ESP1 and determined its three-dimensional structure, which adopts a helical fold stabilized by an intramolecular disulfide bridge with extensive charged patches. We then identified the amino acids involved in the activation of VSNs by a structure-based mutational analysis, revealing that the highly charged surface is crucial for the ESP1 activity. We also demonstrated that ESP1 specifically bound to an extracellular region of V2Rp5 by an in vitro pulldown assay. Based on homology modeling of V2Rp5 using the structure of the metabotropic glutamate receptor, we constructed a docking model of the ESP1-V2Rp5 complex in which the binding interface exhibited good electrostatic complementarity. These experimental results, supported by the molecular docking simulations, reveal that charge-charge interactions determine the specificity of ESP1 binding to V2Rp5 in the large extracellular region characteristic of class C GPCRs. The present study provides insights into the structural basis for the narrowly tuned sensing of mammalian peptide pheromones by class C GPCRs.

Expression and purification of human FROUNT, a common cytosolic regulator of CCR2 and CCR5.

Chemokine receptors play pivotal roles for immune cell recruitment to inflammation sites, in response to chemokine gradients (chemotaxis). The mechanisms of chemokine signaling, especially the initiation of the intracellular signaling cascade, are not well understood. We previously identified a cytoplasmic protein FROUNT, which binds to the C-terminal regions of CCR2 and CCR5 to mediate chemokine signaling. Although large amounts of purified protein are required for detailed biochemical studies and drug screening, no method to produce recombinant FROUNT has been reported. In this study, we developed a method for the production of recombinant human FROUNT. Human FROUNT was successfully expressed in Escherichia coli, as a soluble protein fused to the folding chaperone Trigger Factor, with a cold shock expression system. The purified FROUNT protein displayed CCR2 binding ability without any additional components, as demonstrated by SPR measurements. A gel filtration analysis suggested that FROUNT exists in a homo-oligomeric state. This high-yield method is cost-effective for human FROUNT production. It should be a powerful tool for further biochemical and structural studies to elucidate GPCR regulation and chemokine signaling, and also will contribute to drug development.

Identification of a binding element for the cytoplasmic regulator FROUNT in the membrane-proximal C-terminal region of chemokine receptors CCR2 and CCR5

Chemokine receptors mediate the migration of leucocytes during inflammation. The cytoplasmic protein FROUNT binds to chemokine receptors CCR2 [chemokine (C-C motif) receptor 2] and CCR5, and amplifies chemotactic signals in leucocytes. Although the interaction between FROUNT and chemokine receptors is important for accurate chemotaxis, the interaction mechanism has not been elucidated. In the present study we identified a 16-amino-acid sequence responsible for high-affinity binding of FROUNT at the membrane-proximal C-terminal intracellular region of CCR2 (CCR2 Pro-C) by yeast two-hybrid analysis. Synthesized peptides corresponding to the CCR2 Pro-C sequence directly interacted with FROUNT in vitro. CCR2 Pro-C was predicted to form an amphipathic helix structure. Residues on the hydrophobic side are completely conserved among FROUNT-binding receptors, suggesting that the hydrophobic side is the responsible element for FROUNT binding. The L316T mutation to the hydrophobic side of the predicted helix decreased the affinity for FROUNT. Co-immunoprecipitation assays revealed that the CCR2 L316T mutation diminished the interaction between FROUNT and full-length CCR2 in cells. Furthermore, this mutation impaired the ability of the receptor to mediate chemotaxis. These findings provide the first description of the functional binding element in helix 8 of CCR2 for the cytosolic regulator FROUNT that mediates chemotactic signalling.

Structural basis for the binding of the membrane-proximal C-terminal region of chemokine receptor CCR2 with the cytosolic regulator FROUNT.

The membrane‐proximal C‐terminal region (Pro‐C) is important for the regulation of G‐protein‐coupled receptors (GPCRs), but the binding of the Pro‐C region to a cytosolic regulator has not been structurally analyzed. The chemokine receptor CCR2 is a member of the GPCR superfamily, and the Pro‐C region of CCR2 binds to the cytosolic regulator FROUNT. Studying the interaction between CCR2 Pro‐C and FROUNT at an atomic level provides a basis for understanding the signal transduction mechanism via GPCRs. NOE‐based NMR experiments showed that, when bound to FROUNT, CCR2 Pro‐C adopted a helical conformation, as well as when embedded in dodecylphosphocholine micelles. A comparison of two types of cross‐saturation‐based NMR experiments, applied to a three‐component mixture of Pro‐C, FROUNT and micelles or a two‐component mixture of Pro‐C and micelles, revealed that the hydrophobic binding surface on Pro‐C for FROUNT mostly overlapped with the binding site for micelles, suggesting competitive binding of Pro‐C between FROUNT and micelles. Leu316 was important for both FROUNT and micelle binding. Phe319 was newly identified to be crucial for FROUNT binding, by NMR and mutational analyses. The association and dissociation rates of CCR2 Pro‐C for lipid bilayer biomembranes were faster than those for FROUNT. We previously reported that FROUNT binding to CCR2 is detectable even in unstimulated cells and increases in response to chemokine stimulation. Taken together, these results support a model of CCR2 equilibrium: chemokine binding changes the conformational equilibrium of CCR2 toward the active state, and Pro‐C switches its binding partner from the membrane to FROUNT.

Identification and Preparation of a Novel Chemokine Receptor-Binding Domain in the Cytoplasmic Regulator FROUNT

FROUNT is a cytoplasmic protein that binds to the membrane-proximal C-terminal regions (Pro-Cs) of chemokine receptors, CCR2 and CCR5. The FROUNT–chemokine receptor interactions play a pivotal role in the migration of inflammatory immune cells, indicating the potential of FROUNT as a drug target for inflammatory diseases. To provide the foundation for drug development, structural information of the Pro-C binding region of FROUNT is desired. Here, we defined the novel structural domain (FNT-CB), which mediates the interaction with the chemokine receptors. A recombinant GST-tag-fused FNT-CB protein expression system was constructed. The protein was purified by affinity chromatography and then subjected to in-gel protease digestion of the GST-tag. The released FNT-CB was further purified by anion-exchange and size-exclusion chromatography. Purified FNT-CB adopts a helical structure, as indicated by CD. NMR line-broadening indicated that weak aggregation occurred at sub-millimolar concentrations, but the line-broadening was mitigated by using a deuterated sample in concert with transverse relaxation-optimized spectroscopy. The specific binding of FNT-CB to CCR2 Pro-C was confirmed by the fluorescence-based assay. The improved NMR spectral quality and the retained functional activity of FNT-CB support the feasibility of further structural and functional studies targeted at the anti-inflammatory drug development.

Backbone and side-chain 1H, 15N and 13C assignments of mouse peptide ESP4

A peptide or a small protein released from an exocrine gland or in urine is utilized as a chemosignal that elicits social or reproductive behavior in mice. Recently, we identified the male-specific peptide, exocrine gland-secreting peptide 1 (ESP1), in mouse tear fluids that enhanced female sexual receptive behavior, and determined the three dimensional structure. ESP1 appears to be a member of multigene family that consists of 38 genes in mice, which we call the ESP family. ESP4, a member of the ESP family, is expressed in various exocrine glands, and shows the highest sequence similarity with ESP1. Here, we report the NMR assignments of ESP4 which provides a basis for NMR analyses of this protein. Our results will give insight into structural relationships within the ESP family.

Efficient identification of compounds suppressing protein precipitation via solvent screening using serial deletion mutants of the target protein

The control of protein solubility is a subject of broad interest. Although several solvent screening methods are available to search for compounds that enhance protein solubilization, their performance is influenced by the intrinsic solubility of the tested protein. We now present a method for screening solubilizing compounds, using an array of N‐ or C‐terminal deletion mutants of the protein. A key behind this approach is that such terminal deletions of the protein affect its aggregation propensity. The solubilization activities of trial solvents are individually assessed, based on the number of solubilized mutants. The solubilizing compounds are then identified from the screened solvents. In this study, the C‐terminal chemokine receptor‐binding region of the cytoplasmic protein, FROUNT (FNT‐C), which mediates intracellular signals leading to leukocyte migration, was subjected to the multicomponent screening. In total, 192 solution conditions were tested, using eight terminal deletion mutants of FNT‐C. We identified five solvent conditions that solubilized four or five mutants of FNT‐C, and the compounds in the screened solvents were then, respectively, assessed in terms of their solubilization ability. The best compound for solubilizing FNT‐C was 1,6‐hexanediol. Indeed, 1,6‐hexanediol bound to FNT‐C and suppressed its precipitation, as showed by NMR and dynamic light scattering analyses.

Application of spin-crossover water soluble nanoparticles for use as MRI contrast agents

Water soluble spin-crossover (SCO) iron(II) nanoparticles (NPs) were synthesized by the polyethylene glycol (PEG) coating of [Fe(Htrz)3-3×(NH2trz)3×](BF4)2 (x = 0, 0.1, 0.5 and 1). The NPs with x = 0.1 show gradual SCO behavior over 280–330 K in water. The relaxation times, T1 and T2, were determined and the thermally-responsive T2 values making these NPs a candidate for use as a MRI contrast agent.

1H, 13C and 15N resonance assignments for a chemokine receptor-binding domain of FROUNT, a cytoplasmic regulator of chemotaxis

FROUNT is a cytoplasmic protein that interacts with the membrane-proximal C-terminal regions (Pro-Cs) of the CCR2 and CCR5 chemokine receptors. The interactions between FROUNT and the chemokine receptors play an important role in the migration of inflammatory immune cells. Therefore, FROUNT is a potential drug target for inflammatory diseases. However, the structural basis of the interactions between FROUNT and the chemokine receptors remains to be elucidated. We previously identified the C-terminal region (residues 532–656) of FROUNT as the structural domain responsible for the Pro-C binding, referred to as the chemokine receptor-binding domain (CRBD), and then constructed its mutant, bearing L538E/P612S mutations, with improved NMR spectral quality, referred to as CRBD_LEPS. We now report the main-chain and side-chain 1H, 13C, and 15N resonance assignments of CRBD_LEPS. The NMR signals of CRBD_LEPS were well dispersed and their intensities were uniform on the 1H–15N HSQC spectrum, and thus almost all of the main-chain and side-chain resonances were assigned. This assignment information provides the foundation for NMR studies of the three-dimensional structure of CRBD_LEPS in solution and its interactions with chemokine receptors.