Toshiyuki Kohno

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.

Structural characterization of the BH3-like motif of hepatitis B virus X protein

Hepatitis B virus X protein (HBx) is a multifunctional protein, which is considered to be an essential molecule for viral replication and the development of liver diseases. Recently, it has been demonstrated that HBx can directly interact with Bcl-2 and Bcl-xL through a sequence (termed the BH3-like motif) that is related to the BH3 motif of pro-apoptotic BH3-only proteins. Here, we present the first structural characterization of the HBx BH3-like motif by circular dichroism and NMR spectroscopies. Our results demonstrated that the HBx BH3-like motif has the ability to form an α-helix, and the potential helical region involves residues L108-L134. This is a common characteristic among the BH3 peptides of pro-apoptotic BH3-only proteins, implying that HBx may interact with Bcl-2 and Bcl-xL, by forming an α-helix, similar to the interaction mode of other BH3 peptides with Bcl-2 and Bcl-xL.

A novel neuropilin-1–binding sequence in the human T-cell lymphotropic virus type 1 envelope glycoprotein

Entry of human T-cell lymphotropic virus type 1 (HTLV-1) into host cells is mainly mediated by interactions between the viral envelope glycoprotein surface unit (SU) and three host receptors: glucose transporter type 1, heparin/heparan sulfate proteoglycan, and neuropilin-1 (Nrp1). Here, we analyzed the interaction between HTLV-1 SU and Nrp1 using nuclear magnetic resonance and isothermal titration calorimetry. We found that two SU peptides, residues 85-94 and residues 304-312, bound directly to the Nrp1 b1 domain with affinities of 7.4 and 17.7 μM, respectively. The binding modes of both peptides were almost identical to those observed for Tuftsin and vascular endothelial growth factor A binding to the Nrp1 b1 domain. These results suggest that the C-terminal region of HTLV-1 SU contains a novel site for direct binding of virus to the Nrp1 b1 domain. Our biophysical characterization of the SU peptides may help in developing inhibitors of HTLV-1 entry.

Expression, purification and characterization of hepatitis B virus X protein BH3-like motif-linker-Bcl-xL fusion protein for structural studies

Hepatitis B virus X protein (HBx) is a multifunctional protein that interacts directly with many host proteins. For example, HBx interacts with anti-apoptotic proteins, Bcl-2 and Bcl-xL, through its BH3-like motif, which leads to elevated cytosolic calcium levels, efficient viral DNA replication and the induction of apoptosis. To facilitate sample preparation and perform detailed structural characterization of the complex between HBx and Bcl-xL, we designed and purified a recombinant HBx BH3-like motif-linker-Bcl-xL fusion protein produced in E. coli. The fusion protein was characterized by size exclusion chromatography, circular dichroism and nuclear magnetic resonance experiments. Our results show that the fusion protein is a monomer in aqueous solution, forms a stable intramolecular complex, and likely retains the native conformation of the complex between Bcl-xL and the HBx BH3-like motif. Furthermore, the HBx BH3-like motif of the intramolecular complex forms an α-helix. These observations indicate that the fusion protein should facilitate structural studies aimed at understanding the interaction between HBx and Bcl-xL at the atomic level.

Protein stabilizer, NDSB-195, enhances the dynamics of the β4 -α2 loop of ubiquitin.

Non‐detergent sulfobetaines (NDSBs) are a new group of small, synthetic protein stabilizers, which have advantages over classical compatible osmolytes, such as polyol, amines, and amino acids: they do not increase solution viscosity, unlike polyols, and they are zwitterionic at all pH ranges, unlike amines and amino acids. NDSBs also facilitate the crystallization and refolding of proteins. The mechanism whereby NDSBs exhibit such activities, however, remains elusive. To gain insight into this mechanism, we studied, using nuclear magnetic resonance (NMR), the effects of dimethylethylammonium propane sulfonate (NDSB‐195) on the dynamics of ubiquitin, on which a wealth of information has been accumulated. By analyzing the line width of amide proton resonances and the transverse relaxation rates of nitrogen atoms, we found that NDSB‐195 enhances the microsecond–millisecond dynamics of a β4‐α2 loop of ubiquitin. Although those compounds that enhance protein dynamics are generally considered to destabilize protein molecules, NDSB‐195 enhanced the stability of ubiquitin against guanidium chloride denaturation. Thus, the simultaneous enhancement of stability and flexibility by a single compound can be attained. Copyright

Biochemical characterization of a heterotrimeric Gi-protein activator peptide designed from the junction between the intracellular third loop and sixth transmembrane helix in the m4 muscarinic acetylcholine receptor

Muscarinic acetylcholine receptors (mAChRs) are G-protein coupled receptors (GPCRs) that are activated by acetylcholine released from parasympathetic nerves. The mAChR family comprises 5 subtypes, m1-m5, each of which has a different coupling selectivity for heterotrimeric GTP-binding proteins (G-proteins). m4 mAChR specifically activates the Gi/o family by enhancing the guanine nucleotide exchange factor (GEF) reaction with the Gα subunit through an interaction that occurs via intracellular segments. Here, we report that the m4 mAChR mimetic peptide m4i3c(14)Gly, comprising 14 residues in the junction between the intracellular third loop (i3c) and transmembrane helix VI (TM-VI) extended with a C-terminal glycine residue, presents GEF activity toward the Gi1 α subunit (Gαi1). The m4i3c(14)Gly forms a stable complex with guanine nucleotide-free Gαi1 via three residues in the VTI(L/F) motif, which is conserved within the m2/4 mAChRs. These results suggest that this m4 mAChR mimetic peptide, which comprises the amino acid of the mAChR intracellular segments, is a useful tool for understanding the interaction between GPCRs and G-proteins.

1H, 13C and 15N backbone resonance assignments of the monomeric human M-ficolin fibrinogen-like domain secreted by Brevibacillus choshinensis

M-ficolin, which forms trimer-based multimers, is a pathogen-recognition protein in the innate immune system, and it binds to ligands through its fibrinogen-like (FBG) domain. As the first step toward the elucidation of the molecular basis for pathogen-recognition by the M-ficolin multimers, we assigned the backbone resonances of the monomeric mutant of the M-ficolin FBG domain, recombinantly expressed by Brevibacillus choshinensis. Like the wild-type trimeric FBG domain, the monomeric FBG domain also requires His251, His284 and His297 for the ligand-binding activity, as judged by mutational analyses using zonal affinity chromatography. The secondary structure predicted by the backbone resonance assignments is similar to that of the trimeric FBG domain in the crystal, indicating that the monomeric FBG domain is folded correctly to perform its function.