The tetra-substituted pyrrolidine ring in the title compound has a twisted conformation about the central C—C bond with the N-bound ethylcarboxylate group in an equatorial position and the remaining substituents in axial positions. In the crystal, methyl- and methylene-C—H⋯O(carbonyl) interactions involving all four carbonyl-O atoms lead to supramolecular double-layers. 13H19NO8, is based on a tetra-substituted pyrrolidine ring, which has a twisted conformation about the central C—C bond; the Cm—Ca—Ca—Cme torsion angle is 38.26 (15)° . While the N-bound ethylcarboxylate group occupies an equatorial position, the remaining substituents occupy axial positions. In the crystal, supramolecular double-layers are formed by weak methyl- and methylene-C—H⋯O(carbonyl) interactions involving all four carbonyl-O atoms. The two-dimensional arrays stack along the c axis without directional interactions between them. The Hirshfeld surface is dominated by H⋯H (55.7%) and H⋯C/C⋯H (37.0%) contacts; H⋯H contacts are noted in the inter-double-layer region. The interaction energy calculations point to the importance of the dispersion energy term in the stabilization of the crystal.The title compound, C Specifically, the HCl salt of (I)+ and, finally, hydrochloride formation. In this way, (I)·HCl was obtained in 67% yield , Fig. 1PLATON . The second level, i.e. weaker, of C—H⋯O interactions assemble molecules into a supramolecular layer in the ab plane, Fig. 2b), at separations beyond normally accepted values in PLATON  interaction occurring along the b-axis direction, Fig. 2c). The layer thus formed by these contacts is connected into a double-layer via the methyl-C9—H⋯O5(carbonyl) interactions mentioned above. The double-layers stack along the c axis without directional interactions between them.There are two classes of identifiable non-covalent C—H⋯O interactions occurring in the crystal of (II). As identified in c axis, non-covalent interaction plots  around the specified intermolecular contacts and generates colour-based isosurfaces as detailed in the cited literature. The results, through a three-colour scheme, enable the visualization of contacts as being attractive (blue isosurface), repulsive (red) or otherwise. For the weak interactions in focus, a green isosurface indicates a weakly attractive interaction.Before embarking on a more detailed analysis of the overall molecular packing of (II), in particular of the inter-layer region along the versus sign(λ2)ρ(r) for the three sets of C—H⋯O interactions. The green peaks apparent at density values less than 0.0 a.u. indicate these are weakly attractive interactions.The isosurfaces for three identified C—H⋯O contacts are given in the upper view of Fig. 3dnorm , near the carbonyl-O  and methyl-C—H (H3 and H9B) atoms correspond to the C—H⋯O interactions listed in Table 1b), where the surface around carbonyl-O and methyl-C—H atoms are shown in red  and blue , respectively. Besides the C—H⋯O interactions listed in Table 1A⋯O5 interaction is reflected in the dnorm surface as a faint red spot in Fig. 5a). In addition, short, intra-layer C⋯O contacts with separations 0.01–0.04 Å shorter than the sum of their van der Waals radii, Table 2dnorm surface in Fig. 5b), reflecting the specific influence of the C8, C10 and O1 atoms participating in these contacts.In order to understand further the interactions operating in the crystal of (II), the calculated Hirshfeld surfaces were mapped over the normalized contact distance, de and di diagonal axes, respectively, in Fig. 6a). The individual H⋯H, H⋯O/O⋯H, H⋯C/C⋯H, O⋯O, O⋯C/C⋯O and H⋯N/N⋯H contacts are illustrated in the delineated two-dimensional fingerprint plots (FP) in Fig. 6b)–(g), respectively; the percentage contributions from different interatomic contacts are summarized in Table 3de = di ∼2.4 Å. This short interatomic H⋯H contact involving the methyl-H11C and methylene-H4B atoms, Table 2b axis. Consistent with the C—H⋯O interactions making the major contribution to the directional interactions in the crystal, H⋯O/O⋯H contacts contribute 37.0% to the overall Hirshfeld surface. A distinctive feature in the FP of Fig. 6c), is the two symmetric spikes at de + di ∼2.4 Å. Although H⋯C/C⋯H, O⋯O, O⋯C/C⋯O and H⋯N/N⋯H appear as splash-like distributions of points at de + di ∼3.0 Å, Fig. 6d)–(g), their contributions to the overall Hirshfeld surface are each below than 3.0%. These contacts and the remaining interatomic contacts have only a small effect on the packing, as the sum of their contributions to the overall Hirshfeld surface is less than 8%.The corresponding two-dimensional fingerprint plot for the Hirshfeld surface of (II) is shown with characteristic pseudo-symmetric wings in the upper left and lower right sides of the Eele), polarization (Epol), dispersion (Edis) and exchange-repulsion (Erep) and were scaled as 1.057, 0.740, 0.871 and 0.618, respectively  were calculated using the wave function at the B3LYP/6-31G level of theory. The total energy comprise four terms: electrostatic  with, arguably, the most closely related structure being that of (III) -3,4-bis(acetyloxy)-1-(ethoxycarbonyl)pyrrolidine-2-carboxylic acid  in methanol (1 ml) was cooled to 273–278 K after which an excess of a cold, freshly prepared solution of CH2N2 in ether was added. The mixture was stirred at room temperature for 10 min. Excess CH2N2 was eliminated by purging the balloon with a dry air flow. The purge was collected on a solution of HOAc in MeOH. The reaction solution was concentrated to dryness and the residue was purified by flash column chromatography on silica gel, using a mixture of EtOAc/n-hexane (1:3). Yield: 41.7 mg (quantitative) of (II). Colourless irregular crystals for the X-ray analysis were obtained by the slow evaporation of its n-hexane solution. M.p. 347.6–348.7 K.A solution of : δ = 5.38 ; 5.11 ; 4.51 and 4.42 ; 4.23–4.05 ; 3.91 and 3.87 ; 3.772 and 3.766 ; 3.63 and 3.59 ; 2.10 and 2.09 ; 2.01 and 2.00 ; 1.28 and 1.21 . 1H NMR : δ = 5.62 ; 5.07 and 5.02 ; 4.78 ; 4.55 ; 4.12 and 4.10 ; 4.01–3.88 ; 3.84–3.76 ; 3.65 ; 3.31 and 3.30 ; 1.48 and 1.45 ; 1.43 and 1.42 ; 0.94 and 0.92 . 13C NMR : δ = 169.4; 169.3; 169.2; 168.8; 168.7; 154.8; 154.4; 77.9; 76.9; 74.5; 73.5; 63.6; 63.5; 61.9; 61.7; 52.7; 52.6; 50.6; 50.4; 20.74; 20.70; 20.65; 14.5. Microanalysis calculated for C13H19NO8: C 49.21, H 6.04, N 4.41%. Found: C 48.89, H 6.52, N 4.50%.The Uiso(H) set to 1.2–1.5Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 510.1107/S205698902000701X/hb7916sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S205698902000701X/hb7916Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S205698902000701X/hb7916Isup3.cmlSupporting information file. DOI: 2005478CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The title compound is constructed about a tri-substituted 1,2,3-triazole ring, with the substituent at the C atom flanked by the C and N atoms being a substituted amide group, and with the adjacent C and N atoms bearing phenyl and benzyl groups, respectively. In the crystal, pairwise amide-N—H⋯O(carbonyl) hydrogen bonds give rise to a centrosymmetric dimer. 20H20N4O3, is constructed about a tri-substituted 1,2,3-triazole ring, with the substituent at one C atom flanked by the C and N atoms being a substituted amide group, and with the adjacent C and N atoms bearing phenyl and benzyl groups, respectively; the dihedral angle between the pendant phenyl rings is 81.17 (12)°, indicative of an almost orthogonal disposition. In the crystal, pairwise amide-N—H⋯O(carbonyl) hydrogen bonds lead to a centrosymmetric dimer incorporating methylene-C—H⋯π(benzene) interactions. The dimers are linked into a supramolecular layer in the ab plane via methylene-C—H⋯N(azo) and benzene-C—H⋯O(amide) interactions; the layers stack along the c-axis direction without directional interactions between them. The above-mentioned intermolecular contacts are apparent in the analysis of the calculated Hirshfeld surface, which also provides evidence for short inter-layer H⋯C contacts with a significant dispersion energy contribution.The title compound, C This results in a dihedral angle close to orthogonal for the amide  and carboxylate  residues, i.e. 73.6 (4)°.The molecular structure of (I)a). The molecules in the dimer are linked via a 12-membered {⋯OC3NH}2 synthon and additional stability to the assembly is provided by methylene-C17—H⋯π(benzene) interactions. The dimeric aggregates are connected into a supramolecular layer propagating in the ab plane via methylene-C3—H⋯N2(azo) and benzene-C15—H⋯O1(amide) interactions, Fig. 2b). The layers stack in an …ABAB… pattern along the c axis and inter-digitate to potentially form π–π interactions. However, these are not apparent, Fig. 2c). A more detailed analysis of the interactions occurring in the inter-layer region is provided by an analysis of the calculated Hirshfeld surfaces.The molecular packing in (I)dnorm , i.e. near the amide-H4N and carbonyl-O2 atoms, correspond to the amide-N—H4N⋯O2(carbonyl) hydrogen bond (Table 1b), where the blue  and red  regions are apparent around the amide-H4N and carbonyl-O2 atoms, respectively.In order to probe the interaction between molecules of (I)d Table 1. This hydnorm-mapped Hirshfeld surface in Fig. 4a), with a distance of ∼0.3 Å shorter than the sum of their van der Waals radii, Table 2A) atoms in Fig. 4b) correspond to the inter-layer H7⋯C5, H17A⋯C12 and H12⋯C15 short contacts listed in Table 2dnorm-mapped Hirshfeld surface, this interaction shows up as a distinctive orange ‘pothole’ on the shape-index-mapped Hirshfeld surface, Fig. 5The methylene-C3—H⋯N2(azo) and benzene-C15—H15⋯O1(amide) interactions are observed as faint-red spots on the de and di diagonal axes, respectively, in Fig. 6a). The delineated H⋯H, H⋯C/C⋯H, H⋯O/O⋯H and H⋯N/N⋯H contacts from the overall two-dimensional fingerprint plot are illustrated in Fig. 6b)–(e), respectively. The percentage contributions from different interatomic contacts to the Hirshfeld surface of (I)de = di ∼2.6 Å in Fig. 6b), corresponding to H8⋯H11 ≃2.67 Å  indicating that all H⋯H contacts have long-range characteristics. The H⋯C/C⋯H contacts on the Hirshfeld surface, which contribute 24.9% to the overall surface, Fig. 6c), reflect the C—H⋯π interaction and C⋯H short contacts as discussed above. Consistent with the C—H⋯O and C—H⋯N interactions occurring in the crystal, H⋯O/O⋯H and H⋯N/N⋯H contacts contribute 14.4 and 12.6%, respectively, to the overall Hirshfeld surface. These appear as two sharp symmetric spikes in the fingerprint plots at de + di ≃ 1.9 and 2.4 Å in Fig. 6d) and (e), respectively. The contribution from the other interatomic contacts summarized in Table 2The overall two-dimensional fingerprint plot for the Hirshfeld surface of (I)i.e. the electrostatic (Eele), polarization (Epol), dispersion (Edis) and exchange-repulsion (Erep) energy terms and were calculated with Crystal Explorer 17  hydrogen bond. The next most significant energies of stabilization arise from the methylene-C3—H⋯N2(azo) (dominated by Edis) and benzene-C15—H15⋯O1(amide)  interactions. In terms of energy, the next most significant contributions comes from an interaction in the inter-layer region, namely the H17A⋯C12 contact, Table 4Edis is the dominant contributor. Views of the energy framework diagrams down a axis are shown in Fig. 7Eele of all pairwise interactions sum to −142.9 kJ mol−1, while the total Edis computes to −251.1 kJ mol−1.The pairwise interaction energies between the molecules in the crystal of (I) al. 2017 while EN-phenylamide substituents at the C1- and C2-atoms, respectively et al., 2019Compound (I)Uiso(H) set to 1.2–1.5Ueq(C). The nitrogen-bound H atom was located in a difference Fourier map and refined with N—H = 0.86±0.01 Å, and with Uiso(H) set to 1.2Ueq(N).Crystal data, data collection and structure refinement details are summarized in Table 510.1107/S2056989020007380/hb7921sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989020007380/hb7921Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989020007380/hb7921Isup3.cmlSupporting information file. DOI: 2007664CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In addition, we present (HACA)CON(CA)NH and (HACA)N(CA)CONH, new 4D Hα-start, HN-detect experiments which have two NH dimensions to enhance peak dispersion in a sequential walk through C′, NH and HN, and provide more accurate NH/HN chemical shifts than those that can be obtained from a crowded 1H, 15N-HSQC spectrum. Application of these 4D experiments is demonstrated using BilRI (165 aa), an outer-membrane intrinsically disordered protein from the opportunistic oral pathogen Aggregatibacter actinomycetemcomitans. BilRI amino acid sequence encompasses three very similar repeats with a 13-residue identical stretch in two of them.Resonance assignment of intrinsically disordered proteins is remarkably challenging due to scant chemical shift dispersion arising from conformational heterogeneity. The challenge is even greater if repeating segments are present in the amino acid sequence. To forward unambiguous resonance assignment of intrinsically disordered proteins, we present iHACANCO, HACACON and (HACA)CONCAHA, three HThe online version of this article (10.1007/s10858-020-00299-w) contains supplementary material, which is available to authorized users. Aggregatibacter actinomycetemcomitans is a Gram-negative opportunistic oral pathogen that is linked to periodontitis, infection of tissues supporting the teeth -8 , as demonstrated by its ds Dyson . BilRI ads Dyson , BilRI sds Dyson   was cloned to pET15b vector (Novagen) into the NdeI and XhoI sites. This leads to soluble recombinant BilRI protein with N-terminal His-Tag with a thrombin cleavage site.13C, 15N labeled BilRI was carried out by transforming plasmids into the BL21(DE3) cells. Cells were grown in M9 minimal media, supplemented with 1 g/l of 15NH4Cl and 2 g/l 13C-d-glucose as the sole nitrogen or nitrogen and carbon source, respectively. Cell culture was incubated at 37 °C and temperature was decreased to 16 °C when OD of the cell culture reached 0.4 and protein production was induced with 1 mM IPTG when OD of the cell culture reached 0.6. Cells were further incubated at 16 °C for 16 h and collected by centrifugation. Cells were disrupted with sonication and resulting supernatant was clarified by centrifugation with 30,000×g.Production of Clarified supernatant of BilRI was applied to the 1-mL His GraviTrap column  and the His-Tag was removed by thrombin protease  digestion according to the manufacturer’s instructions. Protease digested mixture was applied to His GraviTrap column. BilRI, without His-Tag, eluted with flow-through. Flow-through was concentrated to volume of 1 ml with Vivaspin 2 concentrator. Concentrated BilRI sample was applied into the Superdex 75 16/60 gel filtration column . Buffer used in gel filtration contained 20 mM sodium phosphate (pH 6.5) and 50 mM NaCl (NMR buffer). Fractions with pure BilRI were pooled and concentrated for NMR studies. The gel filtration was performed by using the ÄKTA Purifier FLPC purification system .15N, 13C labeled protein samples in 5/95% D2O/H2O at pH 6.5. Chemical shifts were referenced to external 2,2,-dimethyl-2-silapentane-5-sulfonic acid (DSS). All data were acquired at 25 °C on a Bruker AVANCE III HD 800 MHz spectrometer, equipped with a TCI 1H/13C/15N cryoprobe. In addition to the new 4D experiments described here, the following experiments were used in the resonance assignment: 2D 1H, 15N-HSQC, constant time 1H, 13C-HSQC, 13C-detected 2D CON NH, HNCO CON, iHA(CA)NCO and (HACA)CON(CA)HA  with accession code 27824.BilRI NMR experiments were acquired using 0.5–1.0 mM 1H, 15N-HSQC spectrum display overwhelming overlap . The CON spectrum, on the other hand, shows remarkably well dispersed signals CON, iHA(CA)NCO and (HACA)CON(CA)HA NH, HNCO and i(HACA)CO(CA)NH NH spectra. Indeed, a precise Cα/Cβ chemical shift for 66% of the residues was obtained, while for Hα, resolved peaks for 87% of the residues were observed .Peaks in the BilRI 2D α overlap problem in 3D HN-detected experiments and to establish direct connectivities between Hα and Cα, and to provide dispersion to solve ambiguities arising from occasional overlap of C′, Hα resonances encountered in the 3D Hα-detected experiments, we devised Hα-detected 4D experiments with Cα as an additional dimension. In addition, we developed and employed new 4D Hα-start, HN-detect experiments which can bridge stretches over single prolines similar to 3D experiments described in Hellman et al. CONCAHA experiments are extensions of their established 3D counterparts → 13Cα(i) transfer, and the density operator immediately after the ϕ3 pulse is described as Hαz(i)Cαz(i) (time point a). Subsequently, the magnetization is transferred to the 13C′ spin followed by the labeling of 13C′ chemical shift in t1 or t2. The relevant density operator after the ϕ2 pulse in all experiments is described as Cαz(i)C′y(i) (time point b). Next, the desired coherence is transferred to the 15N spin of the sequential residue in HACACON and (HACA)CONCAHA experiments, described with the density operator Cαz(i)C′z(i)Ny(i + 1) (time point c). In the iHACANCO experiment, the magnetization is solely transferred to the 15N spin within the residue, described with the density operator Cαz(i)C′z(i)Ny(i) (time point c). After labeling the 15N chemical shifts in t1 (or t2), the magnetization is transferred to the 13Cα coherence after the ϕ4 pulse. The relevant density operators (time point d) are Cαy(i)Nz(i) for iHACANCO, Cαy(i)C′z(i) for HACACON and Cαy(i)Nz(i) for (HACA)CONCAHA schemes. The 13Cα chemical shift is labeled during the t3 period between time points d–e.respectively. All experiments start with the 13Cα(i)→ 15N(i) transfer (Permi αz(i)Cαz(i) coherence (time point a), the 1JCαC′, 1JCαN, 2JCαN and 1JCαCβ couplings are active during the time interval (2TC + 2TA + 2TC′ + TCC) = 52–57 ms, which converts it to the Cαz(i)Ny(i) coherence (time point c). However, during the delay 2TCC, that can be selected to be 0–5 ms based on the relaxation properties of 13Cα spins, only 1JCαCβ is active. This is to maximize the transfer efficiency during the (2TC + 2TA + 2TC′ + TCC) delay. Of note, to avoid chemical shift evolution of 13Cα–13C′ multiple-quantum coherence during 2TA –t2, an additional but opposite frequency labeling period for 13Cα has been implemented in the 2TC′ period. Efficiently, only the chemical shift evolution of 13C′ will take place during t2, but the attainable resolution is limited by 2TC′ (= 2TC + TCC) i.e. t2,max is 19–22 ms, depending on the setting of 2TCC (0–5 ms).While the HACACON Fig. b is the er Permi , 2011. EHA(i), ωCA(i), ωC′(i), ωN(i); ωHA(i), ωCA(i), ωC′(i), ωN(i+1), and ωHA(i), ωCA(i), ωC′(i–1), ωN(i) frequencies, respectively. Given that frequency labeling of 13Cα chemical shifts is implemented in a constant-time manner, without lengthening the actual pulse sequence and incorporating sensitivity enhanced gradient echo in t3, there is no sensitivity loss involved in increasing the dimensionality of these Hα-detected experiments. Hence, the coherence transfer efficiencies provided with the corresponding 3D experiments by Mäntylahti et al.  and two-bond (2JCαN = 7.5 Hz) couplings between backbone 13Cα and 15N spins  for 13Cα (= 100 ms), 13C′ (= 200 ms) and 15N (= 200 ms) spins, we can estimate coherence transfer efficiencies for these experiments in IDPs → 13Cα(i) transfer, 2TNC, during which the 15N magnetization is further modulated by 1JNCδ coupling interaction in prolines.Thus, the 4D iHACANCO, HACACON and (HACA)CONCAHA experiments yield correlations at ωi et al. , 2011 arα-detected with the new 4D spectra: problems associated with multifold overlap and ambiguities in choosing the right sequential connection when using 3D spectra can be surpassed, and the assignment procedure expedited by extending frequency labeling to Cα.Figure α-detected 4D experiments, 76% of the α correlations of were successfully assigned . Considering that some N, C′ resonances are separated by less than 0.05 ppm in the CON spectrum, e.g. corresponding resonances within73/113KDAISE78/118 differ only by 0.03–0.04 ppm, all peaks were not expected to be resolved in spectra with spectral resolutions of 0.11 (15N) and 0.05 (13C) ppm. The Hα, Cα shifts were as ineffective in providing the needed dispersion, the smallest peak separations being comparable, e.g. the 76/116Ile alpha peaks differ by only 0.01 and 0.05 ppm in 1H and 13C, respectively, and those of 77/117Ser even less .With the help of these Hα-detected experiments described above, the 4D HN-detected experiments  , the magnetization is first transferred from 1Hα to 13Cα spin (time point a), and further to either 13C′(i–1) spin in (HACA)CON(CA)NH (density operator Cαz(i–1)C′y(i–1)) or selectively to 15N(i) spin in (HACA)N(CA)CONH (density operator Cαz(i)Ny(i)) at time point b. This is followed by the frequency labeling of 13C′(i–1) and 15N(i) chemical shifts during t1 in (HACA)CON(CA)NH and (HACA)N(CA)CONH experiments, respectively. Next, the desired magnetization is transferred to the sequential 15N spin or intraresidual 13C′ spin, described by the density operators Cαz(i–1)C′z(i–1)Ny(i) and Cαz(i)C′y(i)Nz(i + 1) in (HACA)CON(CA)NH and (HACA)N(CA)CONH, respectively (time point c). Frequency labeling of 15N(i) and 13C′(i) chemical shift takes place in t2. The suppression of sequential 15N pathway in (HACA)N(CA)CONH is accomplished using the intraresidual filtering between time points b and c, and in (HACA)CON(CA)NH between time points c and d. Finally, the chemical shift of 15N(i–1) and 15N(i + 1) chemical shifts are recorded for Cαz(i–1)Ny(i–1) and C′y(i)Nz(i + 1) coherences in (HACA)CON(CA)NH and (HACA)N(CA)CONH between time points d–e.Like in the HHN(i–1), ωN(i–1), ωC′(i–1), ωN(i) and ωHN(i+1), ωN(i+1), ωC′(i), ωN(i) frequencies, respectively. As the magnetization transfer cascade originates from the Hα spin, and the transfer route includes sampling of 15N frequencies of prolines as well, the experiments facilitate assignment of isolated proline residues  together with other residues in IDPs with high sensitivity. Analogously to Hα-detected experiments, under identical conditions (vide supra), the coherence transfer efficiency for both prolines and non-prolines in the 4D (HACA)CON(CA)NH experiment reaches 0.19, if 2TC is set to 28 ms (and 0.15 with 2TC = 6.2 ms) and in the (HACA)N(CA)CONH experiment I ~ 0.20. Hence, both of these experiments are optimal also for the proline assignment in XPX moieties.Hence, the 4D (HACA)CON(CA)NH and (HACA)N(CA)CONH spectra exhibit correlations at the intersection of ω1H, 15N HSQC only 41% of the residues displayed resolved peaks, whereas from the two 4D spectra, accurate amide 1H, 15N shifts were obtained for 85% of BilRI residues. Still overlapping peaks were found for residues 73Lys-80Met, 113Lys-120Met and 153Lys-154Ala in the 71/111/151EVKDAI/VSEKM80/120/160 stretches as well as for residues 103Asp-105Ala, 143Asp-145Ala in the middle of the 99/139KDAKDAAADKM109/149 stretches. Despite the overlap, with the help of the high peak resolution in the CON, it was possible to walk sequentially the whole amino acid sequence with the exception of the first three residues, which are not part of the native BilRI sequence. These residues are missing also from the 1H, 15N HSQC, likely due to fast chemical exchange of their amide proton with the solvent protons. Examples of the resolving power of the additional 1H, 15N HSQC dimension are presented in Fig. In the 71/111EVKDAIVSEKMDAM83/123 and 99/139KDAKDAAADKM109/149 the corresponding peaks are resolved in the CON spectrum. Naturally, the chemical shift differences become small in the middle of the repeats, ~ 0.03–0.04 ppm for both C′ and NH, but the peaks remain, however, distinct. 76/116Ile Cα-Hα is resolved into separate peaks in a high-resolution CT 1H, 13C-HSQC spectrum (t1 acquisition time 51 ms) as well. To have a different chemical shift, the corresponding atoms in the stretches should experience a mutually different structural environment. Future studies will uncover whether this arises from nearest-neighbor effects being effective over five residues or from distinct long-range interactions within the protein.It is interesting to note that throughout the identical stretches N-, 13C′- and Hα-detection approaches CON(CA)NH and (HACA)N(CA)CONH experiments proposed here link the residues flanking a single proline through its 15N chemical shift.During the past several years, several different NMR experiments and assignment schemes have been developed for studying IDPs. Based on the detected spin, these can be categorized to three different classes i.e. HN-detected experiments, the 13C-detection is almost non-susceptible to amide proton exchange with water at high pH. The 13C-detection based experiments for the assignment of IDPs generally apply the ‘CON′ strategy for connecting the neighboring residues in low-complexity regions of IDPs, which is also suitable for the assignment of prolines  attains 0.19, which is comparable to the sensitivity of the (HACA)CON(CA)NH experiment proposed here. However, given that γH/ γC equals 4 and the attainable sensitivity has γ3/2 dependence on the detected spin, the 13C-detection results in an inherent sensitivity loss by a factor of 8 with respect to the 1H-detection. This can partially be redeemed using a 13C-detection optimized probehead and 13C′-detection, which offers higher resolution in the direct detection dimension due to absence of homonuclear couplings. If such a probehead with an inner coil for 13C is not available, this sets limit to the feasible sample concentration (> 0.3–0.5 mM) which might become an issue with some IDPs. On the other hand, homonuclear couling between HN and Hα can be removed in 1H-detected experiments using the homonuclear BASH decoupling scheme , dissolving protein into D2O might be advantageous.Finally, the HN, NH, C′ chemical shifts was obtained for all but the three N-terminal residues, which are not part of the BilRI native sequence. Hα, Cα and Cβ chemical shifts were obtained for 87, 76, and 66% of the residues, respectively. These experiments offer alternative approach for the assignment of IDPs. Hence, they should not be considered superior per se to the existing methods, but as demonstrated here, they have features that may facilitate accomplishing the assignment of challenging IDPs such as BilRI.In all, with the proposed suite of 4D experiments, a complete assignment of backbone Hα-detected and Hα-start, HN-detected 4D experiments that by exploiting chemical shift dispersion brought by an additional Cα or N dimension, facilitate the unambiguous backbone resonance assignment of IDPs in comparison to their established 3D counterparts. The Hα-detected experiments provide versatile strategy for the assignment of proline-rich amino acid sequences and/or proteins at alkali pH, prone to fast chemical exchange with the solvent. The proposed 4D Hα-detected experiments provide information with the most vital chemical shifts i.e. Hα, 13Cα, and 13C′ for conformational restraining in IDPs. The Hα-start, HN-detected (HACA)CON(CA)NH and (HACA)N(CA)CONH experiments offer an alternative assignment protocol for IDPs, by providing connectivities over three sequential residues with the HN-detection that allows for connecting single proline-containing segments of the amino acid sequence. The feasibility of the proposed experiments was experimentally verified on with BilRI, a 165-residue protein, containing three highly similar 40-residue repeats and 12 K-D-A triplet sequences, which represents an extremely challenging case of resonance assignment of IDPs.In this paper, we have presented a suite of HSupplementary file1 (PDF 2150 kb)Below is the link to the electronic supplementary material.


Z′ = 1 form one-dimensional hydrogen-bonded assemblies, those with Z′ = 2 form two-dimensional hydrogen-bonded assemblies, and that with Z′ = 4 forms a three-dimensional hydrogen-bonded assembly. Pseudosymmetry and inversion twinning are apparent when Z′ > 1.Among six closely related 1-halobenzoyl-4-(2-methoxyphenyl)piperazines, those with  N-(2-methoxyphenyl)piperazine. The molecules of 1-(4-fluorobenzoyl)-4-(2-methoxyphenyl)piperazine, C18H19FN2O2 (I), are linked into a chain of rings by a combination of C—H⋯O and C—H⋯π(arene) hydrogen bonds. 1-(4-Chlorobenzoyl)-4-(2-methoxyphenyl)piperazine, C18H19ClN2O2 (II), crystallizes in the space group Pca21 with Z′ = 4 and it exhibits both pseudosymmetry and inversion twinning: a combination of six C—H⋯O and two C—H⋯π(arene) hydrogen bonds generate a three-dimensional assembly. In 1-(4-bromobenzoyl)-4-(2-methoxyphenyl)piperazine, C18H19BrN2O2 (III), which also crystallizes in space group Pca21 but with Z′ = 2, the bromobenzoyl unit in one of the molecules is disordered. Pseudosymmetry and inversion twinning are again present, and a combination of three C—H⋯O and one C—H⋯π(arene) hydrogen bonds generate a two-dimensional assembly. A single C—H⋯O hydrogen bond links the molecules of 1-(4-iodobenzoyl)-4-(2-methoxyphenyl)piperazine, C18H19IN2O2 (IV), into simple chains but in the isomeric 3-iodobenzoyl analogue (V), which crystallizes in space group P212121 with Z′ = 2, a two-dimensional assembly is generated by a combination of four C—H⋯O and two C—H⋯π(arene) hydrogen bonds; pseudosymmetry and inversion twinning are again present. A single C—H⋯O hydrogen bond links the molecules of 1-(2-fluorobenzoyl)-4-(2-methoxyphenyl)piperazine, C18H19FN2O2 (VI), into simple chains. Comparisons are made with the structures of some related compounds.Six 1-halobenzoyl-4-(2-methoxyphenyl)piperazines have been prepared using carbodiimide-mediated coupling reactions between halobenzoic acids and  N-(2-Methoxyphenyl)piperazine (2-MeOPP) has been used as a building block in the synthesis of both 5-HT1A receptor ligands piperazine (4-MeOPP) piperazine and a halogen-substituted benzoic acid.Pca21, but with Z′ values of 2 and 4, respectively; the unit-cell repeat vectors b and c for these two compounds are quite similar, but the a repeat vector for (II)Z′ = 2, but in space group P212121.Despite differing only in the identity of their halogen substituents, no two of compounds (I)–(IV) are isomorphous Figs. 1 ▸ ▸ ▸Z′ > 1, there is considerable flexibility available for the choice of the asymmetric unit: in each case, the asymmetric unit was selected such that the independent molecules in it were linked by C—H⋯O hydrogen bonds s Table 1.P212121 with Z′ = 1, it was not possible to establish the absolute configuration of the molecules in the crystal selected for data collection (see Section 6). In compound (V)cf. Fig. 5Z′ = 2, molecule 1 containing atom Br14 and the major disorder component of molecule 2 containing atom Br24 are related by an approximate, but non-crystallographic b-glide plane at x = ca 0.62 . The arrangement of the molecules in compound (II)1 screw axis along , as also are molecules 2 and 4, containing atoms Cl24 and Cl44 , while molecules 3 and 4 are approximately related by the translation . Compounds (II)For compound (I)x1,Cx2,Cx3,Nx4,Cx5,Cx6) where x = 1 or 2 in (III)x = 1, 2, 3 or 4 in (II)All of the piperazine rings in compounds (I)–(VI) adopt chair type conformations, with values of the ring-puckering angle θ –(IV), the methoxy C atom is close to coplanar with the adjacent aryl ring, with displacements from the plane of the ring ranging from 0.024 (7) Å in molecule 4 of (II)D—H⋯A angles that are significantly less than 140°, as the interaction energies associated with such contacts are likely to be very low, so that these cannot be regarded as structurally significant Z′ = 2 and the aggregation is two-dimensional, and finally (II)Z′ = 4 and the aggregation is three-dimensional.In assessing the intermolecular interactions, we have discounted hydrogen bonds having C(6)   chains are also formed in compounds (IV)1 screw axis along  0) Fig. 7. Simple 5) Fig. 8, while t5) Fig. 8, analogoet al., 1998abet al., 20001 screw axis along   ) Fig. 11 and the ) Fig. 12. Within ) Fig. 12, and the1 screw axis along  to generate a chain running parallel to the [001] direction n Fig. 14 and chaiet al., 2019Pbca, all with Z′ = 1 –(VI) with those of some analogous compounds. In the structure of 1-(2-fluorobenzoyl)-4-(4-methoxyphenyl)piperazine (VII), which is isomeric with compound (VI)N-(2-methoxyphenyl)piperazine  in N,N-dimethylformamide (5 ml) was then added to each of the mixtures prepared as above, followed by stirring that was continued overnight at room temperature. When the reactions were confirmed to be complete using thin-layer chromatography, each mixture was then quenched with water (10 ml) and extracted with ethyl acetate (20 ml). Each organic fraction was separated and washed successively with an aqueous hydrochloric acid solution (1 M), a saturated solution of sodium hydrogencarbonate and then with brine. The organic phases were dried over anhydrous sodium sulfate and the solvent was then removed under reduced pressure. The resulting solid products were then crystallized from acetone–ethyl acetate  for (I)v/v) solvent mixtures for (II)–(VI): m.p. (I)All reagents were commercially available and all were used as received. For the synthesis of compounds (I)–(VI), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide , 1-hydroxybenzotriazole  and triethylamine  were added to a solution of the appropriately substituted benzoic acid (0.52 mmol) in methanol (10 ml), thus 4-fluorobenzoic acid (73 mg) for (I)3) and 0.97 Å (CH2), and with Uiso(H) = kUeq(C), where k = 1.5 for the methyl groups, which were allowed to rotate but not to tilt, and 1.2 for all other H atoms. For the minor disorder component in (III)x parameter /[(I+) + (I−)], means that the absolute structure is indeterminate  calculated using 1164 quotients; for (III)x = 0.300 (6) calculated using 1164 quotients; and for (V)x = 0.456 (12) calculated using 1728 quotients. The structure of (I)3 and centred close to  and ; however, examination of the refined structure using SQUEEZE  global, I, II, III, IV, V, VI. DOI: 10.1107/S2056989020015649/hb7954Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989020015649/hb7954IIsup3.hklStructure factors: contains datablock(s) II. DOI: 10.1107/S2056989020015649/hb7954IIIsup4.hklStructure factors: contains datablock(s) III. DOI: 10.1107/S2056989020015649/hb7954IVsup5.hklStructure factors: contains datablock(s) IV. DOI: 10.1107/S2056989020015649/hb7954Vsup6.hklStructure factors: contains datablock(s) V. DOI: 10.1107/S2056989020015649/hb7954VIsup7.hklStructure factors: contains datablock(s) VI. DOI: Click here for additional data file.10.1107/S2056989020015649/hb7954Isup8.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989020015649/hb7954IIsup9.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989020015649/hb7954IIIsup10.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989020015649/hb7954IVsup11.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989020015649/hb7954Vsup12.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989020015649/hb7954VIsup13.cmlSupporting information file. DOI: 2046985, 2046984, 2046983, 2046982, 2046981, 2046980CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Pulsatilla chinensis (Bunge) Regel (Ranunculaceae), has many pharmacological uses, but its effect on cancer cell metabolism is still unclear.α-Hederin, a potent bioactive compound of To elucidate the role of α-hederin in the glucose metabolism of lung cancer cells.Cell Counting Kit 8 and colony formation assays were employed to assess the antiproliferative effects of α-hederin. Glucose uptake, ATP generation, and lactate production were measured. Glycolysis-related proteins were detected using western blotting, and a sirtuin 6 (SIRT6) inhibitor was used to verify A549 cell proliferation. Sixty male BALB/c nude mice were divided into normal control, 5-FU (25 mg/kg), and α-hederin (5 and 10 mg/kg) groups to assess the antitumor effect for 32 days. Glycolysis-related protein expression was evaluated using immunohistochemical analysis.50 = 13.75 μM), NCI-H460 (IC50 = 17.57 μM), and NCI-H292 (IC50 = 18.04 μM) proliferation; inhibited glucose uptake and ATP generation; and reduced lactate production. Furthermore, α-hederin (10 and 15 μM) markedly inhibited hexokinase 2, glucose transporter 1, pyruvate kinase M2, lactate dehydrogenase A, monocarboxylate transporter, c-Myc, hypoxia-inducible factor-1α, and activated SIRT6 protein expression. Using a SIRT6 inhibitor, we demonstrated that α-hederin inhibits glycolysis by activating SIRT6. A tumour xenograft mouse model of lung cancer confirmed that α-hederin (5 and 10 mg/kg) inhibits lung cancer growth by inhibiting glycolysis in vivo.α-Hederin inhibited A549 (ICα-Hederin inhibits A549 cell growth by inhibiting SIRT6-dependent glycolysis. α-Hederin might serve as a potential agent to suppress cancer. In recent years, the incidence of lung cancer has been increasing. Lung cancer is the deadliest form of cancer and is responsible for about a quarter of all cancer deaths  Regel (Ranunculaceae), which is used for its cooling and detoxifying abilities, has been reported to have many pharmacological characteristics such as being antimalarial and antibacterial  and SIRT6 inhibitor OSS_128167 were purchased from MedChemExpress . Hexokinase 2 (HK2), glucose transporter 1 (GLUT1), pyruvate kinase M2 (PKM2), monocarboxylate transporter (MCT4), lactate dehydrogenase A (LDHA), c-Myc, p53, hypoxia-inducible factor-1α (HIF-1α), protein kinase B (Akt), and p-Akt antibodies were purchased from Abcam . SIRT6 antibody was purchased from Proteintech .α-Hederin (purity 99%) was provided by Chengdu Push Bio-Technology Co. Ltd.  and the cell count kit 8 (CCK8) was purchased from Dojindo Laboratories . RPMI-1640 and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from HyClone Laboratories . Foetal bovine serum (FBS) was obtained from Biological Industries . 2-Deoxy-2 incubator at 37 °C.Human NSCLC A549 cells and human lung cancer cells NCI-H460 and NCI-H292 (lymph node metastasis) were provided by the Stem Cell Bank of the Chinese Academy of Sciences. NCI-H460 and NCI-H292 cells were cultured in RPMI-1640 medium containing 10% FBS, and A549 cells were cultured in DMEM containing 10% FBS in a 5% COThe effect of α-hederin on the proliferation of A549, NCI-H292, and NCI-H460 cells was assessed using the CCK8 assay. Cell suspensions were seeded into 96-well plates at 5000 cells/well. After incubation for 24 h, the cells were treated with α-hederin  for 48 h. Subsequently, 10 μL of CCK8 was added to each well and the plate was incubated for 1 h. Absorbance was measured at 450 nm using a SpectraMax® i3 Multi-mode Microplate Reader . The inhibition rate was calculated using the following formula:The effect of α-hederin on cell proliferation was detected via colony formation assay. A549 cells were seeded into 6-well plates (500 cells/well) and incubated for 24 h, after which the cells were treated with 5, 10, or 15 μM α-hederin for 48 h. The cells were then cultured in DMEM containing 10% FBS for 10 days, followed by fixation with 4% formaldehyde and staining with 0.5% crystal violet. The number of colonies was quantified.5 cells/well) and incubated for 24 h. Then DMEM containing 10% FBS and 0, 5, 10, or 15 μM α-hederin were seeded into the 6-well plates, the plates were incubated for 24 h, and culture media were collected. Glucose, lactate, and ATP levels were assayed using a kit  following the manufacturer’s instructions. Absorbance was determined using a microplate reader, and the amount of glucose uptake, lactate generated, and ATP levels in the cells per protein mass were determined.A549 cells were cultured in 6-well plates (2 × 106 cells/dish), some of which were treated with α-hederin for 24 h. The cells were then lysed in RIPA buffer containing protease and phosphatase inhibitors . Proteins were separated out using SDS-PAGE based on molecular weight and blotted onto a polyvinylidene fluoride membrane (Thermo Fisher Scientific). Bands were incubated with the primary antibodies HK2 (1:2000), GLUT1 , PKM2 (1:1000), MCT4 (1:1000), LDHA (1:1000), c-Myc (1:1000), p53 (1:1000), HIF-1α (1:1000), Akt (1:1000), p-Akt (1:1000), and SIRT6 (1:1000) overnight at 4 °C and measured using a ChemiDoc Imaging System with Image Lab Software version 4.1 .For western blot analysis, A549 cells were placed in 6-cm dishes  was detached using scissors. A small incision was made on the lateral abdomen of the recipient animals and a small piece of tumour tissue was implanted into the right armpit using unhooked ophthalmic tweezers. The blank group of mice was injected with culture medium when the tumour volume reached 100 mm3. Then the mice were randomly assigned to five groups and treated intraperitoneally with α-hederin (10 or 5 mg/kg), 5-FU (25 mg/kg), or a vehicle every other day. The mice were euthanized after 32 days of treatment and tumour volume (length × width2 × 0.5236) and body weight were determined. Finally, the tumours were fixed in 4% paraformaldehyde.Six-week-old male BALB/c nude mice  were used as an A549 xenograft tumour mouse model. The mice were housed in a pathogen-free environment at the institutional animal care facility at 21 °C and relative humidity of 55% with free access to food and water. The mice were injected with 5 × 10The procedures for the care and use of the animals were approved by the experimental animal ethics committee of Jiangxi University of Traditional Chinese Medicine (JZLLSC2018-0053), and all applicable institutional and governmental regulations concerning the ethical use of animals were followed.Tumour tissues were embedded in paraffin and cut into 5-μm sections using a microtome . Following the instructions of the manufacturer , tumour sections were incubated with GLUT1 (1:400), HK2 (1:400), PKM2 (1:30), LDHA (1:400), MCT4 (1:100), HIF-1α (1:200), c-Myc (1:100), and SIRT6 (1:100) antibodies overnight at 4 °C. Tumours were taken from each group, 3 sections were taken from each tumour, and 6 fields were chosen from each slice. The results were calculated using the mean integrated optical density and analysed using Image-Pro Plus 6.0 .t-test was used for statistical analyses between two independent groups. All tests were performed using IBM SPSS Statistics 19 , and p < 0.05 was considered significant.Significant differences between individual groups were determined by comparison using one-way ANOVA. A 50 of α-hederin in NSCLC A549, NCI-H460, and NCI-H292 was 13.75, 17.57, and 18.04 μM, respectively. α-Hederin exhibited a superior inhibitory effect on A549 cells, which subsequently were used for experiments (Our CCK8 results showed that α-hederin had significant inhibitory effects on the proliferation of human lung cancer cells. The ICeriments .p < 0.05), with 15 μM α-hederin having the best inhibitory effect  (p < 0.05) (p < 0.05) , glucose consumption by A549 cells treated with 2DG and 10 and 15 μM α-hederin was significantly lower ( < 0.01) . Compare < 0.05) . Compare < 0.05) .GLUT1 transports extracellular glucose into the cell, which then generates lactate and ATP via the catalysis of enzymes, including HK2, PKM2, and LDHA. Lactate and ATP are used in tumour cell proliferation. Finally, MCT4 transports intracellular lactate out of the cell. Western blot results showed that compared to 0 µM α-hederin, 10 and 15 μM α-hederin significantly reduced the expression of GLUT1, HK2, PKM2, LDHA, and MCT4 .c-Myc, HIF-1α, Akt, and p53 have distinct effects on proteins involved in glycolysis in tumour cells. The results of our study showed that each α-hederin dose had no significant effect on the expression of glycolysis regulators Akt and p53 protein in tumour cells, while the expression of c-Myc and HIF-1α was significantly downregulated in each α-hederin group compared to the 0 μM α-hederin group . ExpressOur results showed that SIRT6 expression was significantly upregulated in each group compared to that in the control . After tWe examined the inhibitory effect of α-hederin combined with the SIRT6 inhibitor OSS_128167 on the proliferation of A549 cells. CCK8 results showed that compared to the inhibitory effect of α-hederin alone, that of α-hederin combined with OSS_128167 was significantly decreased .p < 0.05). The average tumour weights of 10 (0.39 g) and 5 mg/kg (0.49 g) α-hederin groups were significantly lower than that of the control (0.65 g), and the inhibitory rates of 10 and 5 mg/kg α-hederin were 40% and 24%  (p < 0.05)  . In addi < 0.05) .Figure 8.in vivo. IHC of GLUT1, HK2, PKM2, LDHA, MCT4, HIF-1α, c-Myc and SIRT6 in tumour tissues (200×). The results showed a remarkable decrease in expression of GLUT1, HK2, PKM2, LDHA, MCT4, HIF-1α, c-Myc and SIRT6 in tumours treatment with 5 and 10 mg/kg α-hederin compared to controls. *p < 0.05, **p < 0.01 as compared to the Normal control group. Mean ± S.E.M. n = 6.α-Hederin suppresses expression of glycolysis-related proteins in vivo and in vitro and significantly reduced glucose uptake, reduced lactate generation, and reduced ATP levels in the NSCLC A549 cells. The inhibitory mechanism of α-hederin is mediated by SIRT6, and α-hederin regulates c-Myc and HIF-1α, which reduce the key glycolytic enzymes GLUT1, HK2, PKM2, LDHA, and MCT4.This study assessed the inhibitory effect of α-hederin on tumour cells and measured the effects of glucose, lactate, and intracellular ATP in cell culture medium on glycolysis in A549 cells. Expression of GLUT1, HK2, PKM2, LDHA, MCT4, and glycolytic regulators c-Myc, HIF-1α, p53, Akt, and SIRT6 was detected using western blot assays to determine the inhibitory mechanism of α-hederin on NSCLC A549 cells. We established an NSCLC A549 allograft transplantation tumour model to investigate the effect of α-hederin on xenograft tumours in mice by evaluating tumour volume and weight. In addition, immunohistochemistry was used to detect the expression of glycolysis-related proteins. The results showed that α-hederin inhibited NSCLC A549 In the 1920s, Otto Warburg discovered that glycolysis is the primary source of energy for cancer cells, even when oxygen levels are sufficient (Koppenol et al. in vitro and in vivo.GLUTs are involved in the entry of glucose into cells. GLU1 plays an important role in GLUTs Kapoor . HK is tp53 is a tumour suppressor gene. The p53 protein reprograms energy metabolism to negatively regulate cell glycolysis by promoting mitochondrial oxidative phosphorylation and inhibiting glycolysis (Matoba et al. Myc gene family of oncogenes and is associated with a wide variety of tumour development. c-Myc promotes glucose absorption by upregulating GLUT1 (Osthus et al. In cancer cells, GLUT1, HK2, PKM2, LDHA, MCT4, and other glycolysis-related proteins directly affect the glycolytic process. However, these key enzymes are regulated by relevant regulatory factors. Akt plays an important role in cell energy metabolism and may enhance aerobic glycolysis in tumour cells (Polivka and Janku SIRT6 plays a key regulatory role in gene transcription, metabolism, maintenance of genomic stability, and integrity of telomeres, thus regulating the occurrence and development of diabetes, obesity, heart disease, cancer, and other diseases. SIRT6 inhibited the activity of the transcription factor HIF-1α and glucose oxidation and glycolysis via the citric acid cycle (Shun et al. 50 of α-hederin in A549 cells was 13.75 μM and 10 mg/kg α-hederin had the strongest inhibitory effect on tumour growth in xenografted nude mice. The underlying mechanisms with respect to our findings are that α-hederin activates SIRT6 expression, inhibits the expression of glycolytic regulatory factors HIF-1α and c-Myc, and suppresses the expression of glycolytic proteins (In our study, α-hederin inhibited the growth of NSCLC A549 cells by suppressing glycolysis. The ICproteins .


Polychlorinated bisphenols (PCBs) continue to contaminate food chains globally where they concentrate in tissues and disrupt the endocrine systems of species throughout the ecosphere. Hydroxylated PCBs (OH-PCBs) are major PCB metabolites and high-affinity inhibitors of human estrogen sulfotransferase (SULT1E1), which sulfonates estrogens and thus prevents them from binding to and activating their receptors. OH-PCB inhibition of SULT1E1 is believed to contribute significantly to PCB-based endocrine disruption. Here, for the first time, the molecular basis of OH-PCB inhibition of SULT1E1 is revealed in a structure of SULT1E1 in complex with OH-PCB1  and its substrates, estradiol (E2), and PAP (3’-phosphoadenosine-5-phosphosulfate). OH-PCB1 prevents catalysis by intercalating between E2 and catalytic residues and establishes a new E2-binding site whose E2 affinity and positioning are greater than and competitive with those of the reactive-binding pocket. Such complexes have not been observed previously and offer a novel template for the design of high-affinity inhibitors. Mutating residues in direct contact with OH-PCB weaken its affinity without compromising the enzyme’s catalytic parameters. These OH-PCB resistant mutants were used in stable transfectant studies to demonstrate that OH-PCBs regulate estrogen receptors in cultured human cell lines by binding the OH-PCB binding pocket of SULT1E1. SULTs catalyze regiospecific transfer of the sulfuryl moiety .i OH-PCB values range as low as ∼100 pM (Hydroxylated polychlorinated biphenyls (OH-PCBs) are the most potent known SULT1E1 inhibitors—K ∼100 pM . OH-PCBs ∼100 pM . PCBs ha ∼100 pM , 23, 24, ∼100 pM , 26. OH- ∼100 pM , and PCB ∼100 pM . Approxi ∼100 pM , when th ∼100 pM . Remedia ∼100 pM .To better understand the molecular basis of OH-PCB action, and with the intent to use the findings as a template for the design and synthesis of potent SULT1E1-specfic inhibitors, we determined the solution structure of SULT1E1 bound to PAP, estradiol (E2), and OH-PCB1 . Structui ∼ 400 PM (The OH-PCBs used in our studies are shown in ∼ 400 PM ) and it ∼ 400 PM , 32, PCB∼ 400 PM .via mutagenesis.The effects of an unpaired spin-label electron on the solution NMR spectrum of a bound ligand in rapid exchange , 35, 36 α RMSF be ≤1.0 Å and that the RMSF of the modeled spin-label nitroxyl-oxygen be ∼6.0 Å, suggesting unfettered motion.Spin label attachment sites were selected from SULT1E1 backbone regions predicted to be stable by MD evaluation of the fully equilibrated E⋅PAPS⋅E2 scaffold. Surface residues that are solvent exposed and well isolated from the catalytic machinery were selected from the stable regions using the criterion that they be separated to maximize the spin-label paramagnetic field coverage of the protein surface. Finally, all insertion points satisfied the criteria that their Cvia PCR-based mutagenesis. Prior to creating spin-label attachment constructs, reactive Cys was removed from the native scaffold. Only one of the four native SULT1E1 Cys  reacts with DTNB  of each spin-labeled mutant were determined  to evaluate whether the catalytic integrity of the constructs had been compromised by the insertions and/or spin label attachments. The results, compiled in Spin labels were attached to the SULT1E1 scaffold at Cys residues that were site-specifically incorporated ith DTNB , and mutith DTNB  and the 1H-NMR peaks, thus allowing distances to be determined, regardless of where the ligand binds. The spin labels used in the current study are presented in r) between the unpaired electron and nucleus is given by the following equation r2 values (R2obs) for the bound species can be obtained from the slopes of Solution-Phase-Linewidth versus Fraction-Ligand-Bound plots   and the protein (the diamagnetic contribution). Interspin distance calculations (Equation 2), which is obtained by subtracting the diamagnetic contribution from R2obs. The diamagnetic contribution is given by the slopes of LW-versus-FB plots constructed using control constructs in which the spin-label PROXYL-moiety  is replaced by the cyclohexyl-group  , 44. Theyl-group  . The 12 interspin distances needed to triangulate the protons associated with the four resonances were obtained from LW-versus-FB plots  to move within an ellipsoid whose centerpoint is given by the intersection of the three NMR distance vectors that position it and whose principal axes lengths correspond to the standard errors (±1 σ) of the distance measurements. The restoring force (applied using distance_restraints in GROMACS) drives a proton toward the center of its ellipsoid if it lies outside of the ellipsoid surface -weighted distance restrains, which were applied simultaneously to the 12 distances that constrain the four OH-PCB1 positions. Docking simulations were run long enough to allow structures to achieve equilibrium. The results of ten simulations were analyzed using g_cluster in GROMACS. Only a single structural cluster (≤2.0 Å RMSD) was detected. The resulting structures were virtually identical and are overlain in NMR triangulation distances were used in conjunction with MD docking simulations , 46 to o surface , 46. Givurements , 38, 47,A. OH-PCB1 is situated in a highly hydrophobic pocket and is in direct contact with the three residues highlighted in blue . The OH-PCB1 benzyl rings are sandwiched on one side by the rings of P75 and P80 and on the other by those of E2. The ring plane of Y239 contacts edges of both OH-PCB1 and E2. Remarkably, the binding of OH-PCB1 establishes a new E2-binding site in which it prevents catalysis by intercalating between E2 and the catalytic machinery. The structure reveals how an inhibitor (OH-PCB1) that sterically prevents a substrate (E2) from binding its active-site pocket can be transformed from a competitor to an allostere whose effects cannot be diminished by increasing substrate concentrations.The structure of OH-PCB1 bound to its binding site in the E⋅PAPS⋅E2⋅OH-PCB1 complex is presented in Figure 5via H-bonding with His107  and vertically, which enhances 3-OH reactivity h His107  and reach His107  and is vh His107 .i OH-PCB1) can be used to validate the structure. To assess whether mutagenizing OH-PCB1 binding-site residues influences the catalytic functions of SULT1E1, the effects of such mutations on the initial-rate parameters (Km and kcat) of 1-hydroxypyrene . In contrast, the mutations had pronounced effects on Ki OH-PCB1. Mutating Y239, which contacts an edge of OH-PCB1, causes a 5.7-fold decrease in affinity, while mutating residues that sandwich the inhibitor (F75 and F80) result in larger decreases . The effect of the F75/F80 double mutation, 113-fold, is within error equal to the product of the individual mutations, suggesting that these residues operate largely independently. These findings fully support the structure seen in Figure 5A, and confirm that the OH-PCB1-binding and catalytic functions of SULT1E1 are indeed separable.In the absence of inhibitor, the olive residues are in direct contact with E2, and the blue residues lie slightly beyond E2-contact distance, suggesting that their contribution to substrate binding and catalysis might be slight. If the OH-PCB1-binding and catalytic functions of the site are separable, the effects of mutating the OH-PCB1 binding site on the OH-PCB1 inhibition constant  werubstrate . OH-PCB1ubstrate , 44 indivia ligand-binding induced changes in SULT1E1 intrinsic fluorescence . Notably, PAP binding does not alter the energetics of OH-PCB1/E2 interactions—OH-PCB1 affinities for E⋅E2 and E⋅E2⋅PAP are experimentally indistinguishable (62 ± 2 and 66 ± 3 nM).To better understand the inhibition mechanism, interactions between OH-PCBs and reactants were probed using equilibrium binding studies. OH-PCB-binding titrations were performed at saturating reactant concentrations and monitored nce see, . OH-PCB1C and D, and m E2 = 5 nM , respectively (2 = 5 nM ).d concentrations of E2 ; similarly, E2 affinity did not vary at 100- and 1000 × Kd concentrations OH-PCB2 —see, A–C and d ratios they require.To assess whether OH-PCBs can add directly to the E⋅E2 complex, and vice versa, the order of binding OH-PCB and E2 was determined. To do so, the affinity of each ligand was assessed at two saturating and tenfold different concentrations of the partner ligand. If binding is ordered, the ligand’s affinity will appear to increase tenfold as the partner draws it onto the enzyme. If instead, binding is random, the ligand affinity will not vary with the change in partner concentration. The affinity of OH-PCB1 did not vary at 32- and 320 × Km and kcat) were determined and proved to be identical within error  were comparable with those reported for human mammary epithelial cells  SULT1E1 were constructed see,  from Ishror see, . A (-) Sror see, . Extractn−1 mg−1 ) and 52-via alkaline phosphatase activity and the resulting titrations are presented in Figure 10A. Endogenous alkaline phosphatase levels are coordinated with ER activation and measured via para-nitrophenyl phosphate hydrolysis (50 of E2 is 80-fold higher in the WT (red dots) and MT (blue dots) strains, which express SULT1E1, than in the control strain (black dots), which does not  used in the OH-PCB-titrations shown in Panels B and C. The OH-PCB1 and OH-PCB2 ER-activation patterns are similar across the three strains. OH-PCBs do not affect activation in the control strain; hence, any effects on activation in the WT and MT strains are likely linked to SULT1E1 expression. OH-PCB effects on the WT strain are pronounced—activation begins at background and increases to a maximum comparable with that associated with the control strain. OH-PCB1 and OH-PCB2 EC50 values  are similar to their Ki values (60 and 0.50 nM), suggesting that the plots are reporting increases in E2 activity as SULT1E1 is inhibited. To establish that the OH-PCB effects are due to binding at the OH-PCB-binding site seen in Figure 2A, activation in the MT strain was tested. As is evident, activation in the MT strain remains near background throughout the entire OH-PCB concentration range with the exception of slight elevation at the highest OH-PCB concentrations, which may be due to relatively weak inhibition of the mutant and/or low-affinity OH-PCB activation of the receptor , neomycin, pcDNA 3.1, para-nitrophenyl-phosphate (pNpp), tris(hydroxymethyl) amino-methane (Tris) base, phenylmethylsulfonyl fluoride (PMSF), radioimmunoprecipitation (RIPA) buffer, and tetramethylsilane (TMS) were purchased from Fisher Scientific. 4ʹ-OH-2,6-dichlorobiphenol and 4-OH-3,3',4',5-tetrachloro-biphenol were purchased from ChemTik. BGII and Gibson Assembly mutagenesis kits were purchased from New England Biolabs. D2O and D2-cholorform (>99%) were purchased from Cambridge Isotope Laboratories. Glutathione- and nickel-chelating resins were obtained from GE Healthcare. Lipofectamine and Opti-MEM were purchased from EMD Millipore Corporation. 17-Beta-estradiol was purchased from Steraloids. 3H-estradiol was purchased from PerkinElmer. Competent E. coli (BL21(DE3)) was purchased from Novagen. PAPS and PAP are synthesized in-house according to published protocols  (DTNB), dithiothreitol (DTT), ethylenediamine-tetraacetic acid (EDTA), L-glutathione (reduced), 1-hydroxypyrene (1-HP), Ishikawa cells, imidazole, isopropyl-thio-β-D-galactopyranoside (IPTG), lysozyme, 3-maleimido-PROXYL , N-cyclo-hexylmaleimide, pepstatin A, potassium phosphate, and 2,2,2-trichloroethanol (TCE) were the highest grade available from Sigma. Ampicillin, fetal bovine serum (FBS), KOH, LB media, MgClrotocols . PAPS anhttp://www.GROMACS.org under the GROMACS General Public License (GPL)  , 46, 60..edu.au/ , 62. A G.edu.au/ .E. coli codon-optimized SULT1E1 coding region inserted into the PreScission Protease cleavage site of a triple-tagged pGEX-6P expression vector containing an -His/GST/MBP tag ) harboring an SULT1E1 pGEX-6P expression plasmid containing a His/GST/MBP triple tag was grown at 37 °C in LB medium , pepstatin A (1.5 μM), lysozyme (0.10 mg/ml), EDTA (2.0 mM), KCl (400 mM), K2PO4 (50 mM), pH 7.5). The suspension was sonicated, centrifuged , and the supernatant was collected. MgCl2 (5.0 mM) was added to the supernatant to chelate EDTA before passing it through a Chelating Sepharose Fast Flow column charged with Ni2+. The column was washed (imidazole (10 mM), KCl (400 mM), and KPO4 (50 mM), pH 7.5), and the enzyme was eluted (imidazole (250 mM), KCl (400 mM), and KPO4 (50 mM), pH 7.5) and loaded directly onto a Glutathione Sepharose column. The GST column was washed (DTT (2.0 mM), KCl (400 mM), and KPO4 (50 mM), pH 7.5), and the tagged enzyme was then eluted (reduced glutathione (10 mM), DTT (2.0 mM), KCl (400 mM), and Tris (100 mM), pH 8.0). The fusion protein was digested overnight at 4 °C using PreScission Protease and passed through a GST column to remove the tag. The protein was ≥95% pure as judged by SDS-PAGE using 2,2,2-trichloroethanol (TCE) treated gels, and its concentration was determined by UV absorbance . The pr4 (pH 7.4), 25 °C ± 2 deg. C. PAP was added to enhance enzyme stability. The reactions were monitored by using DTNB to measure unreacted cysteine and were considered complete when >98% of the cysteine had reacted (∼3 h).Labels  were added in 20-fold excess over reactive cysteine to a solution containing SULT1E1 construct (50 μM active sites), 0.50 mM PAP, and 50 mM KPOvia ligand-induced changes in the intrinsic fluorescence of the enzyme  (d) SULT1E1 , PAP , E2 , KPO4 (50 mM), pH 7.5, 25 °C ± 2 deg. C. Titrations were performed in triplicate and the averaged data were least-squares fit to the following quadratic, single-site binding model: Io − ΔI∙(([L]+Etot + Kd) − [([L]+Etot + Kd)2 − (4∙[L]∙Etot)]1/2)/2∙Etot, where Io and ΔI represent fluorescent intensity at zero and infinite [L].The binding of inhibitors to WT and mutant SULT1E1 was monitored m) to a solution containing SULT1E1 , 1-HP , and KPO4 (50 mM), pH 7.5, 25 °C ± 2 deg. C. Reaction progress was monitored via the fluorescence change associated with 1-HP sulfonation -weighted least-squares fitting of the averaged data in double-reciprocal space  . Ins 1/[S]) , 67.m), OH-PCBs were added (0.20–20 × Ki), and SULT1E1 was 0.20 nM (active sites). Ki was determined by least-squares fitting to a noncompetitive inhibition model , KPO4 (50 mM), pH 7.5, 25 °C ± 2 deg. C. Reactions were initiated by addition of PAPS  and quenched after 90 to 180 s with KOH . The reaction mixture was diluted tenfold with an E2 (10 μM), KPO4 (50 mM), pH 7.5 solution, and brought to pH 7.0 with HCl (6.0 N). The reaction was then mixed (1:1 v/v) with neat chloroform and centrifuged . The aqueous layer was removed, chloroform was extracted twice more, and radioactivity in the aqueous layer was then determined by liquid scintillation counting. Reactions were performed in triplicate and Km and kcat were obtained by (1/v4)-weighted least-squares fitting of the data in double-reciprocal space   .2-choloroform (≥99%), 25 °C ± 1 deg. C, spectral windows: 0 to 14 (proton spectra) and 0 to 200 ppm (carbon spectra). Peak assignments were made using 1H-13C Heteronuclear Single Quantum Coherence (HSQC) (NMR experiments were performed using a Bruker 600 MHz spectrometer equipped with a TCI H/F-cryogenic probe at 298 °K. 1D-proton and 1D-carbon data collection conditions: OH-PCB1 (500 μM), TMS (0.50 mM), De (HSQC)  and Hetee (HSQC) .d), E2 (4.0 μM + [SULT1E1active sites]), PAP , KPO4 (50 mM), pD 7.4, 25 °C ± 1 deg. C. Peak widths were obtained by fitting to a Lorentzian distribution using NMRdraw , OH-PCB1  and lipofectamine (2.5 units ml−1). After 24 h at 37 °C ± 2 deg. C, cells were washed (3×) with PBS before adding growth medium containing neomycin (400 μg ml−1) to select transfectants. Selective growth medium was replenished every 48 h until single colonies could be isolated (∼3–6 weeks). Single colonies were transferred using trypsin digest (0.05%) to 12-well plates and grown at 37 °C ± 2 deg. C to confluence for further experimentation and storage.pcDNA 3.1 constructs harboring either the SULT1E1 WT or double mutant (F75A/F80A) coding regions were generated using Gibson Assembly . The vecg for 10 min, 25 °C) and the supernatant was collected, assayed, flash-frozen, with liquid nitrogen, and stored at −80 °C. SULT1E1 activity was stable following freeze/thaw. Extract protein concentrations were determined using the Bradford assay  was added in lieu of pure enzyme.Transfectants were grown at 37 °C ± 2 deg. C to 60 to 70% confluency in 12-well plates, washed (3×) with PBS (25 °C), and lysed using RIPA buffer (0.50 ml) . Lysate rd assay  and SULTTransfected Ishikawa cells were grown at 37 °C ± 2 deg. C to 60 to 70% confluency in MEM containing 10% v/v FBS. Cells were then transferred to 96-well tissue culture plates and grown at 37 °C ± 2 deg. C to 80% confluency in MEM containing 10% v/v charcoal filtered FBS. The plate was then washed with PBS and grown at 37 °C ± 2 deg. C for 24 h in MEM without FBS. Estradiol (0–3.0 μM) and OH-PCB (0–1.0 μM) solubilized in neat DMSO were added and cells were incubated at 37 °C ± 2 deg. C for 5 days. The final concentrations of DMSO were ≤0.10%. Cells were then washed twice with PBS before adding pNpp (5.0 mM) in PBS . Followitom.leyh@einsteinmed.org. All SULT1E1⋅E2⋅ PAPS⋅OH-PCB1 spin-label directed docking models and the NIH SAVE v5.0 structure validation report are available for download at www.model.archive.org (accession no. ma-xnec7).All data and materials are available upon request at The authors declare that they have no conflicts of interest with the contents of this article.


We identified a novel heterozygous variant, Bβp.Pro234Leu (fibrinogen Tokorozawa), which was suspected to be associated with hypofibrinogenemia. Therefore, we analyzed the assembly and secretion of this fibrinogen using Chinese hamster ovary (CHO) cells. To determine the impact on the synthesis and secretion of fibrinogen of the Bβp.P234L and γp.G242E substitutions, we established recombinant variant fibrinogen-producing CHO cell lines. Synthesis and secretion analyses were performed using an enzyme-linked immunosorbent assay (ELISA) and immunoblotting analysis with the established cell lines. In addition, we performed fibrin polymerization using purified plasma fibrinogen and in-silico analysis. Both Bβp.P234L and γp.G242E impaired the secretion and synthesis of fibrinogen. Moreover, immunoblotting analysis elucidated the mobility migration of the Bβγ complex in Bβp.P234L. On the other hand, the fibrin polymerization of fibrinogen Tokorozawa was similar to that of normal fibrinogen. In-silico analysis revealed that the Bβp.P234 residue is located in the contact region between the Bβ and γ chains and contacts γp.G242 residue. The present study demonstrated that the Bβp.P234L substitution resulted in hypofibrinogenemia by decreasing the assembly and secretion of fibrinogen. Therefore, there is a possibility that substitutions in the contact region between the Bβ and γ chains impact the assembly and secretion of fibrinogen. FGA, FGB, and FGG, are clustered on chromosome 4, and these genes translate into polypeptides for the pre-pro-Aα chain (644 residues), pre-pro-Bβ chain (491 residues), and pre-pro-γ chain (437 residues) [2, which is then secreted into the circulation [Fibrinogen is a 340 kDa plasma glycoprotein involved in hemostasis by forming fibrin . The hum chain 49 residues chain 49 residuesesidues) . Each ch chain 49 residuesUp to 800 congenital fibrinogen disorders (CFDs) have been listed on the Groupe d’études sur l’hémostase et la thrombose (GEHT) database . CFDs arIn this study, we identified a novel amino acid substitution associated with hypofibrinogenemia. Furthermore, we analyzed the impact of this substitution on the assembly and secretion of fibrinogen using a Chinese hamster ovary (CHO) cell line.The proposita of fibrinogen Tokorozawa was a 36-year-old Japanese female subjected to surgery due to miscarriage at 12 weeks of gestation. The laboratory data before the operation showed that her functional fibrinogen level was 1.36 g/L, and her prothrombin time-international normalized ratio was 1.13. However, fibrin/fibrinogen degradation products, D-dimer, factor VII, factor XIII, and the activated partial thromboplastin time (APTT) were within normal ranges. During the operation, excessive bleeding occurred and 3 g of fibrinogen concentration was administered. After that, she improved. Although she and her family members had not experienced any episodes of abnormal bleeding or thrombosis, she was referred to Shinshu University Hospital to investigate the cause of lower fibrinogen concentration.For the Tokorozawa proposita, the functional and immunological plasma fibrinogen concentrations (reference range: 1.80–3.50 g/L) were 1.24 and 1.16 g/L, respectively, and the functional/immunological ratio was 1.07 (0.90–1.10). The prothrombin time (PT) and APTT were 11.1 s (10.8–13.2 s) and 28.9 s (23.0–38.0 s), respectively. This proposita was diagnosed with suspected hypofibrinogenemia.FGB exon 4 c.701C>T, resulting in the substitution of Leu (CTT) for Pro (CCT) at the Bβ204 residue (mature protein: BβP204L) or Bβp.234 residue (native protein: Bβp.P234L)  . Any othWe established a Bβp.P234L fibrinogen-producing CHO cell line and a γp.G242E fibrinogen-producing CHO cell line, since the Bβp.P234 residue contacts the γp.G242 residue. We will explain the contact between Bβp.P234 and γp.G242 residues in detail in a later section.n = 8, median (interquartile range)) were 0.33 (0.20–0.43) μg/mL in the culture media and 0.36 (0.31–0.39) μg/mL in cell lysates, resulting in a ratio of culture media to cell lysates (M/C ratio) of 0.95 (0.73–1.18). In the culture media of the variant fibrinogen-producing CHO cells, the fibrinogen concentrations of Bβp.P234L  μg/mL) and γp.G242E  μg/mL) were significantly lower than that of WT (The fibrinogen concentrations of wild-type (WT)  showed the typical pattern for Aα, Bβ, and γ chains on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in reducing conditions. In addition, three fibrinogen bands , low molecular weight-fibrinogen , or LMW’-fibrinogen (270 kDa) were identified in non-reducing conditions a,b. The 30min) were obtained from the turbidity curves. The polymerization curve of fibrinogen Tokorozawa (heterozygous Bβp.P234L) was similar to that of normal fibrinogen. Moreover, there were no significant differences between normal fibrinogen and fibrinogen Tokorozawa in either lag time : 2.4 ± 0.20 min vs. fibrinogen Tokorozawa: 2.5 ± 0.10 min), Vmax (8.9 ± 0.29 × 10−4/s vs. 9.1 ± 0.59 × 10−4/s), or ΔAbs30min (0.280 ± 0.002 vs. 0.262 ± 0.013). Therefore, the coagulation function of fibrinogen Tokorozawa was normal.We performed thrombin-catalyzed fibrin polymerization at room temperature using purified plasma fibrinogen, and turbidity curves were obtained as shown in Gallus gallus fibrinogen and Petromyzon marinus fibrinogen revealed that the former fibrinogen Bβp.P208 and latter fibrinogen Bβp.P223 (these residues correspond to human fibrinogen Bβp.P234) also contact the residues corresponding to human fibrinogen γp.L205, γp.G242, γp.H243, and γp.L244  . From these structures, the residues that contact the Bβp.P234 residue were γp.G242, γp.H243, and γp.L244. In addition, for 2FFD and 1LTJ, the γp.L205 residue also contacted Bβp.P234 a. Accordγp.L242) a.To determine the highly conserved residues, we performed the multiple amino acid sequence alignment. As shown in To reveal the impact on the Bβγ complex structure from each mutation, we performed the in-silico molecular analysis, and these results are shown in We identified the novel heterozygous variant, Bβp.P234L, which was designated as fibrinogen Tokorozawa. We considered that this substitution caused hypofibrinogenemia since the proposita’s immunological plasma fibrinogen concentration was low but the functional/immunological ratio was normal. Therefore, we performed thrombin-catalyzed fibrin polymerization using purified plasma fibrinogen and analyzed the secretion and synthesis of variant fibrinogen using CHO cells. The results of thrombin-catalyzed fibrin polymerization revealed that the coagulation function of fibrinogen Tokorozawa was normal. Moreover, in the analysis of secretion and synthesis, although the variant Bβ chain was synthesized normally in the cell, a small amount of Bβp.P234L fibrinogen was assembled in the cell, and just a limited proportion was secreted into the culture medium. In other words, the Bβp.P234L substitution impaired both the assembly and secretion of fibrinogen. Therefore, we demonstrated that the Bβp.P234L substitution caused hypofibrinogenemia.In addition, since the Bβp.P234 residue was in contact with the γp.G242 residue, we established a recombinant γp.G242E fibrinogen-producing CHO cell line and also analyzed the secretion and synthesis of fibrinogen. Two cases of γp.G242E fibrinogen have been reported as fibrinogen Caracas and fibrinogen French Basque, and this substitution was also associated with hypofibrinogenemia ,16. MoreThe Bβp.P234 residue is located on the Bβ chain surface area where the contact with the γ chain occurs. Moreover, there are interchain disulfide bonds, Aαp.C180-γp.C161, Aαp.C184-Bβp.C223, and Bβp.C227-γp.C165, around the C-terminal side contact region between the Bβ and γ chains . TherefoG. gallus and P. marinus have been registered in the PDB data bank among these species except humans, we confirmed that the region of these species’ fibrinogen structure corresponded to the region around the human fibrinogen Bβp.P234 residue. Notably, both residues of G. gallus and P. marinus corresponding to the human fibrinogen Bβp.P234 residue were contacted with the same residues of human fibrinogen. In addition, these γ chain residues were also highly conserved in various animals. Therefore, it is considered that these residues play an important role in proper assembly into the Bβγ complex and secretion as a six-chain dimer.The causative mutations for quantitative disorders in the Bβ and γ chains were clustered in each highly conserved C-terminal globular domain . TherefoThis study was approved by the Ethics Review Board of Shinshu University School of Medicine . After the informed consent had been obtained from the proposita, blood samples were collected for biochemical and genetic analyses.Blood collection from the proposita and plasma separation were performed as described previously . PT, APTFGA, FGB, and FGG, polymerase chain reaction (PCR) primers were designed as described previously [To amplify all exons and exon–intron boundaries in eviously . DNA waseviously .TTGTGGTGTCTGGC-3′, reverse: 5′-GCCAGACACCACAAGAATATTGCAACTGACAGTG-3′). Mutated plasmids were co-transfected with histidinol selection plasmid into CHO cells that expressed the WT human fibrinogen Aα chain and γ chain, and colonies were selected on histidinol  [AACATCTGTCTCCTACTGGCACAACAG-3′, reverse: 5′-CTGTTGTGCCAGTAGGAGACAGATGTTCAAATCCTTC-3′) and CHO cells that expressed the WT human fibrinogen Aα chain and Bβ chain as described above. As a control, we used eight clones of the WT human fibrinogen-producing CHO cell established as described previously [A Bβp.P234L substitution was introduced into the fibrinogen Bβ chain expression vector pMLP-Bβ plasmid , which contained WT Bβ cDNA, using a QuikChange II Site-Directed Mutagenesis Kit  and the primers  . Moreoveeviously .6 cells) and the conditioned media was harvested 1 day after reaching confluence (6–8 days after seeding). Then, from the same dish, CHO cells were harvested. These were washed three times, and dissolved in a 50 mM Tris-HCl pH 8.0 buffer added with 0.1% IGEPAL CA-630  and 10 mM phenylmethylsulfonyl fluoride . Fibrinogen concentrations in these samples were measured using ELISA as previously described [Culture media and cell lysates for ELISA were prepared as follows. CHO cells were cultured to confluence in 60-mm dishes  was performed using immunoaffinity chromatography with an IF-1 monoclonal antibody -conjugated Sepharose 4B column, and purified fibrinogen concentrations were determined as described previously . The pur2: 1 mM. Three parameters: Lag time, Vmax, and ΔAbs30min, were obtained from the turbidity curves as described previously [Turbidity curves of fibrin polymerization were recorded at 350 nm using a UV-1280 . Recordings were performed as described previously . The fineviously . Reactiohttp://www.protein.osaka-u.ac.jp/rcsfp/supracryst/suzuki/jpxtal/Katsutani/InterfaceResidues.py). Moreover, using the fibrinogen structure of pheasant  ,23,24,25ID: 1M1J ) and lamID: 1LWU ), the γ ID: 1LWU .Homo sapiens, Bβ chain ID: P02675 and γ chain ID: P02679-2), chimpanzee , monkey , boar , bovine , horse , goat , sheep , rabbit , cat , dog , mouse , rat , pheasant , lizard , turtle , frog , zebrafish , and lamprey .We performed the multiple amino acid sequence alignment of the fibrinogen Bβ chain or γ chain using the Clustal Omega program , and theThe fibrinogen structure models with Bβp.P234L or γp.G242E substitutions were prepared as follows. One hundred models of each mutation were prepared using Modeller9.24  and eitht-test was used to compare the three parameters of thrombin-catalyzed fibrin polymerization. A difference was considered to be significant when the p-value was <0.05. Box-and-whisker plots and dot plots were prepared using the R software (version 3.5.1) [The statistical analysis was performed using the EZR software . The Krun 3.5.1) .We identified a novel amino acid substitution in the Bβ chain, Bβp.P234L, and designated this as fibrinogen Tokorozawa. This substitution was associated with hypofibrinogenemia by impairing the assembly and secretion of fibrinogen. Moreover, the γp.G242E substitution, which is located in the contact region of the Bβp.P234 residue, also inhibited the assembly and secretion of fibrinogen. Therefore, there is a possibility that the contact region between the Bβ and γ chains has a principal role in the assembly and secretion of fibrinogen.