Structural Insights into the Interaction between the C-Terminal-Deleted BH3-like Motif Peptide of Hepatitis B Virus X Protein and Bcl-xL.

Hepatitis B virus X protein (HBx) plays a crucial role in the development of hepatocellular carcinoma (HCC) associated with hepatitis B virus (HBV) infection. The full-length HBx protein interacts with Bcl-xL and is involved in the HBV replication and cell death processes. The three hydrophobic residues Trp120, Leu123, and Ile127 of the HBx BH3-like motif are essential for the Bcl-xL-binding. On the other hand, various lengths of C-terminal-truncated HBx mutants are frequently detected in HCC tissues, and these mutants, rather than the full-length HBx, appear to be responsible for HCC development. Notably, the region spanning residues 1-120 of HBx [HBx(1 and 120)] has been strongly associated with an increased risk of HCC development. However, the mode of interaction between HBx(1-120) and Bcl-xL remains unclear. HBx(1-120) possesses only Trp120 among the three hydrophobic residues essential for the Bcl-xL-binding. To elucidate this interaction mode, we employed a C-terminal-deleted HBx BH3-like motif peptide composed of residues 101-120. Here, we present the NMR complex structure of Bcl-xL and HBx(101-120). Our results demonstrate that HBx(101-120) binds to Bcl-xL in a weaker manner. Considering the high expression of Bcl-xL in HCC cells, this weak interaction, in conjunction with the overexpression of Bcl-xL in HCC cells, may potentially contribute to HCC development through the interaction between C-terminal-truncated HBx and Bcl-xL.

Author Correction: A spatial similarity of stereochemical environments formed by amino acid residues defines a common epitope of two non-homologous proteins.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A spatial similarity of stereochemical environments formed by amino acid residues defines a common epitope of two non-homologous proteins.

It is critical for development of high-quality antibodies in research and diagnostics to predict accurately their cross-reactivities with "off-target" molecules, which potentially induce false results. Herein, we report a good example of such a cross-reactivity for an off-target due to a stereochemical environment of epitopes, which does not simply depend on amino acid sequences. We found that significant subpopulation of a polyclonal peptide antibody against Bcnt (Bucentaur) (anti-BCNT-C antibody) cross-reacted with a completely different protein, glutamine synthetase (GS), and identified four amino acids, GYFE, in its C-terminal region as the core amino acids for the cross-reaction. Consistent with this finding, the anti-BCNT-C antibody strongly recognized endogenously and exogenously expressed GS in tissues and cultured cells by Western blotting and immunohistochemistry. Furthermore, we elucidated that the cross-reaction is caused by a spatial similarity between the stereochemical environments formed by amino acid residues, including the GYFE of GS and the GYIE of Bcnt, rather than by their primary sequences. These results suggest it is critical to comprehensively analyze antibody interactions with target molecules including off-targets with special attention to the physicochemical environments of epitope-paratope interfaces to decrease the risk of false interpretations of results using antibodies in science and clinical applications.

NMR characterization of the interaction between Bcl-xL and the BH3-like motif of hepatitis B virus X protein.

Hepatitis B virus X protein (HBx) possesses a BH3-like motif that directly interacts with the anti-apoptotic proteins, Bcl-2 and Bcl-xL. Here we report the interaction between the HBx BH3-like motif and Bcl-xL, as revealed by nuclear magnetic resonance spectroscopy. Our results showed that this motif binds to the common BH3-binding hydrophobic groove on the surface of Bcl-xL, with a binding affinity of 89 µM. Furthermore, we examined the role of the tryptophan residue (Trp120) in this motif in Bcl-xL binding using three mutants. The W120A mutant showed weaker binding affinity (294 µM) to Bcl-xL, whereas the W120L and W120F mutants exhibited almost equivalent binding affinity to the wild-type. These results indicate that the bulky hydrophobic residues are important for Bcl-xL binding. The findings will be helpful in understanding the apoptosis networks between viral proteins and host factors.

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 ß4 -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 ß4 -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.

Mammalian Bcnt/Cfdp1, a potential epigenetic factor characterized by an acidic stretch in the disordered N-terminal and Ser250 phosphorylation in the conserved C-terminal regions.

The BCNT (Bucentaur) superfamily is classified by an uncharacteristic conserved sequence of ∼80 amino acids (aa) at the C-terminus, BCNT-C (the conserved C-terminal region of Bcnt/Cfdp1). Whereas the yeast Swc5 and Drosophila Yeti homologues play crucial roles in chromatin remodelling organization, mammalian Bcnt/Cfdp1 (craniofacial developmental protein 1) remains poorly understood. The protein, which lacks cysteine, is largely disordered and comprises an acidic N-terminal region, a lysine/glutamic acid/proline-rich 40 aa sequence and BCNT-C. It shows complex mobility on SDS/PAGE at ∼50 kDa, whereas its calculated molecular mass is ∼33 kDa. To characterize this mobility discrepancy and the effects of post-translational modifications (PTMs), we expressed various deleted His-Bcnt in E. coli and HEK cells and found that an acidic stretch in the N-terminal region is a main cause of the gel shift. Exogenous BCNT/CFDP1 constitutively expressed in HEK clones appears as a doublet at 49 and 47 kDa, slower than the protein expressed in Escherichia coli but faster than the endogenous protein on SDS/PAGE. Among seven in vivo phosphorylation sites, Ser(250), which resides in a region between disordered and ordered regions in BCNT-C, is heavily phosphorylated and detected predominantly in the 49 kDa band. Together with experiments involving treatment with phosphatases and Ser(250) substitutions, the results indicate that the complex behaviour of Bcnt/Cfdp1 on SDS/PAGE is caused mainly by an acidic stretch in the N-terminal region and Ser(250) phosphorylation in BCNT-C. Furthermore, Bcnt/Cfdp1 is acetylated in vitro by CREB-binding protein (CBP) and four lysine residues including Lys(268) in BCNT-C are also acetylated in vivo, revealing a protein regulated at multiple levels.

Biochemical characterization of a heterotrimeric G(i)-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.

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.

Commissioning of 6 MV medical linac for dynamic MLC-based IMRT on Monte Carlo code GEANT4.

Monte Carlo simulation is the most accurate tool for calculating dose distributions. In particular, the Electron Gamma shower computer code has been widely used for multi-purpose research in radiotherapy, but Monte Carlo GEANT4 (GEometry ANd Tracking) is rare for radiotherapy with photon beams and needs to be verified further under various irradiation conditions, particularly multi-leaf collimator-based intensity-modulated radiation therapy (MLC-based IMRT). In this study, GEANT4 was used for modeling of a 6 MV linac for dynamic MLC-based IMRT. To verify the modeling of our linac, we compared the calculated data with the measured depth-dose for a 10 × 10 cm(2) field and the measured dose profile for a 35 × 35 cm(2) field. Moreover, 120 MLCs were modeled on the GEANT4. Five tests of MLC modeling were performed: (I) MLC transmission, (II) MLC transmission profile including intra- and inter-leaf leakage, (III) tongue-and-groove leakage, (IV) a simple field with different field sizes by use of MLC and (V) a dynamic MLC-based IMRT field. For all tests, the calculations were compared with measurements of an ionization chamber and radiographic film. The calculations agreed with the measurements: MLC transmissions by calculations and measurements were 1.76 ± 0.01 and 1.87 ± 0.01 %, respectively. In gamma evaluation method (3 %/3 mm), the pass rates of the (IV) and (V) tests were 98.5 and 97.0 %, respectively. Furthermore, tongue-and-groove leakage could be calculated by GEANT4, and it agreed with the film measurements. The procedure of commissioning of dynamic MLC-based IMRT for GEANT4 is proposed in this study.

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.

Solution structure of kurtoxin: a gating modifier selective for Cav3 voltage-gated Ca(2+) channels.

Kurtoxin is a 63-amino acid polypeptide isolated from the venom of the South African scorpion Parabuthus transvaalicus. It is the first and only peptide ligand known to interact with Cav3 (T-type) voltage-gated Ca(2+) channels with high affinity and to modify the voltage-dependent gating of these channels. Here we describe the nuclear magnetic resonance (NMR) solution structure of kurtoxin determined using two- and three-dimensional NMR spectroscopy with dynamical simulated annealing calculations. The molecular structure of the toxin was highly similar to those of scorpion α-toxins and contained an α-helix, three ß-strands, and several turns stabilized by four disulfide bonds. This so-called "cysteine-stabilized α-helix and ß-sheet (CSαß)" motif is found in a number of functionally varied small proteins. A detailed comparison of the backbone structure of kurtoxin with those of the scorpion α-toxins revealed that three regions [first long loop (Asp(8)-Ile(15)), ß-hairpin loop (Gly(39)-Leu(42)), and C-terminal segment (Arg(57)-Ala(63))] in kurtoxin significantly differ from the corresponding regions in scorpion α-toxins, suggesting that these regions may be important for interacting with Cav3 (T-type) Ca(2+) channels. In addition, the surface profile of kurtoxin shows a larger and more focused electropositive patch along with a larger hydrophobic surface compared to those seen on scorpion α-toxins. These distinct surface properties of kurtoxin could explain its binding to Cav3 (T-type) voltage-gated Ca(2+) channels.

Microdosimetric study on influence of low energy photons on relative biological effectiveness under therapeutic conditions using 6 MV linac.

PURPOSE: Microdosimetry has been developed for the evaluation of radiation quality, and single-event dose-mean lineal energy y(D) is well-used to represent the radiation quality. In this study, the changes of the relative biological effectiveness (RBE) values under the therapeutic conditions using a 6 MV linac were investigated with a microdosimetric method. METHODS: The y(D) values under the various irradiation conditions for x-rays from a 6 MV linac were measured with a tissue-equivalent proportional counter (TEPC) at an extremely low dose rate of a few tens of microGy/min by decreasing the gun grid voltage of the linac. According to the microdosimetric kinetic model (MK model), the RBE(MK) values for cell killing of the human salivary gland (HSG) tumor cells can be derived if the y(D) values are obtained from TEPC measurements. The Monte Carlo code GEANT4 was also used to calculate the photon energy distributions and to investigate the changes of the y(D) values under the various conditions. RESULTS: The changes of the y(D) values were less than approximately 10% when the field size and the depth in a phantom varied. However, in the measurements perpendicular to a central beam axis, large changes were observed between the y(D) values inside the field and those outside the field. The maximum increase of approximately 50% in the y(D) value outside the field was obtained compared with those inside the field. The GEANT4 calculations showed that there existed a large relative number of low energy photons outside of the field as compared with inside of the field. The percentages of the photon fluences below 200 keV outside the field were approximately 40% against approximately 8% inside the field. By using the MK model, the field size and the depth dependence of the RBEMK values were less than approximately 2% inside the field. However, the RBEMK values outside the field were 6.6% higher than those inside the field. CONCLUSIONS: The increase of the RBE(MK) values by 6.6% outside the field was observed. This increase is caused by the change of the photon energy distributions, especially the increase of the relative number of low energy photons outside the field.

Synthesis of unnatural alkaloid scaffolds by exploiting plant polyketide synthase.

HsPKS1 from Huperzia serrata is a type III polyketide synthase (PKS) with remarkable substrate tolerance and catalytic potential. Here we present the synthesis of unnatural unique polyketide-alkaloid hybrid molecules by exploiting the enzyme reaction using precursor-directed and structure-based approaches. HsPKS1 produced novel pyridoisoindole (or benzopyridoisoindole) with the 6.5.6-fused (or 6.6.5.6-fused) ring system by the condensation of 2-carbamoylbenzoyl-CoA (or 3-carbamoyl-2-naphthoyl-CoA), a synthetic nitrogen-containing nonphysiological starter substrate, with two molecules of malonyl-CoA. The structure-based S348G mutant not only extended the product chain length but also altered the cyclization mechanism to produce a biologically active, ring-expanded 6.7.6-fused dibenzoazepine, by the condensation of 2-carbamoylbenzoyl-CoA with three malonyl-CoAs. Thus, the basic nitrogen atom and the structure-based mutagenesis enabled additional C─C and C─N bond formation to generate the novel polyketide-alkaloid scaffold.