Charge neutralization and ß-elimination cleavage mechanism of family 42 L-rhamnose-α-1,4-D-glucuronate lyase revealed using neutron crystallography.

Gum arabic (GA) is widely used as an emulsion stabilizer and edible coating and consists of a complex carbohydrate moiety with a rhamnosyl-glucuronate group capping the non-reducing ends. Enzymes that can specifically cleave the glycosidic chains of GA and modify their properties are valuable for structural analysis and industrial application. Cryogenic X-ray crystal structure of GA-specific L-rhamnose-α-1,4-D-glucuronate lyase from Fusarium oxysporum (FoRham1), belonging to the polysaccharide lyase (PL) family 42, has been previously reported. To determine the specific reaction mechanism based on its hydrogen-containing enzyme structure, we performed joint X-ray/neutron crystallography of FoRham1. Large crystals were grown in the presence of L-rhamnose (a reaction product), and neutron and X-ray diffraction datasets were collected at room temperature at 1.80 and 1.25 Å resolutions, respectively. The active site contained L-rhamnose and acetate, the latter being a partial analog of glucuronate. Incomplete H/D exchange between Arg166 and acetate suggested that a strong salt-bridge interaction was maintained. Doubly deuterated His105 and deuterated Tyr150 supported the interaction between Arg166 and the acetate. The unique hydrogen-rich environment functions as a charge neutralizer for glucuronate and stabilizes the oxyanion intermediate. The NE2 atom of His85 was deprotonated and formed a hydrogen bond with the deuterated O1 hydroxy of L-rhamnose, indicating the function of His85 as the base/acid catalyst for bond cleavage via ß-elimination. Asp83 functions as a pivot between the two catalytic histidine residues by bridging them. This His-His-Asp structural motif is conserved in the PL 24, 25, and 42 families.

A comparison of opioid dose between home palliative care and hospital palliative care.

OBJECTIVE: While opioids are a key part of palliative care, few studies have evaluated opioid demand in the home care context. This study aims to compare opioid usage in home care and hospital care settings. METHODS: This cross-sectional study retrospectively recruited patients receiving palliative care in home care and hospital settings, between November 2018 and October 2020. Opioid prescriptions were standardized to oral morphine equivalent (OME) doses at 7 and 14 days prior to death and analyzed. Additional analysis performed multivariable linear regression on the outcome of OME at 7 days, adjusting for medical setting and confounders in patients with opioid prescriptions. RESULTS: After 21 exclusions, 209 patients (48 home care and 161 hospital care) were eligible for analysis. The home care group had a higher mean age (74.8 years) and Palliative Prognosis Score (50), than the hospital group (70.1 and 40, respectively). Mean OME at 7 and 14 days before death was numerically higher in the home care group (72.8 mg/day and 53.0 mg/day, respectively) than the hospital care group (57.7 mg/day and 35.7 mg/day). Student's t-test produced p-values of 0.49 and 0.32, and the Wilcoxon rank sum test found p-values of 0.24 and 0.11 at 7 and 14 days, respectively. Multivariable regression analysis of the home care group found mean OME of 40.7 mg/day; 95% confidence interval [-0.62, 82.0 (mg/day)], p = 0.06. Additional analysis found a p-value of 0.06 for medical setting. CONCLUSIONS: We did not find a statistically significant difference in opioid use between home care and hospital care. However, the numerically higher rate of use in the home care group suggests that further research is warranted.

Resveratrol Derivatives Inhibit Transthyretin Fibrillization: Structural Insights into the Interactions between Resveratrol Derivatives and Transthyretin.

Hereditary ATTR amyloidosis is a disease caused by the deposition of amyloid fibrils formed by mutated transthyretin (TTR), a protein that binds to thyroid hormone in the serum, in the organs. The development of a small molecule that binds to and stabilizes TTR is a promising strategy for the treatment of ATTR amyloidosis. In the present study, we demonstrated that the resveratrol derivatives including pterostilbene available as a dietary supplement inhibit the fibrillization of V30M-TTR to the same extent as the approved drug tafamidis. Furthermore, based on a thermodynamic and X-ray crystallographic analysis, the binding of the resveratrol derivative to TTR was shown to be enthalpy-driven, with the binding enthalpy being acquired by hydrogen bonding to S117. Moreover, direct observation of hydrogen atoms by neutron crystallography provided details of the hydrogen bond network by S117 and emphasized the importance of the CH···π interaction by L110 in the ligand binding.

New insights into the oxidation process from neutron and X-ray crystal structures of an O2-sensitive [NiFe]-hydrogenase.

[NiFe]-hydrogenase from Desulfovibrio vulgaris Miyazaki F is an O2-sensitive enzyme that is inactivated in the presence of O2 but the oxidized enzyme can recover its catalytic activity by reacting with H2 under anaerobic conditions. Here, we report the first neutron structure of [NiFe]-hydrogenase in its oxidized state, determined at a resolution of 2.20 Å. This resolution allowed us to reinvestigate the structure of the oxidized active site and to observe the positions of protons in several short hydrogen bonds. X-ray anomalous scattering data revealed that a part of the Ni ion is dissociated from the active site Ni-Fe complex and forms a new square-planar Ni complex, accompanied by rearrangement of the coordinated thiolate ligands. One of the thiolate Sγ atoms is oxidized to a sulfenate anion but remains attached to the Ni ion, which was evaluated by quantum chemical calculations. These results suggest that the square-planar complex can be generated by the attack of reactive oxygen species derived from O2, as distinct from one-electron oxidation leading to a conventional oxidized form of the Ni-Fe complex. Another major finding of this neutron structure analysis is that the Cys17S thiolate Sγ atom coordinating to the proximal Fe-S cluster forms an unusual hydrogen bond with the main-chain amide N atom of Gly19S with a distance of 3.25 Å, where the amide proton appears to be delocalized between the donor and acceptor atoms. This observation provides insight into the contribution of the coordinated thiolate ligands to the redox reaction of the Fe-S cluster.

Rafoxanide, a salicylanilide anthelmintic, interacts with human plasma protein transthyretin.

Transthyretin (TTR) is a carrier protein for thyroid hormone thyroxine (T4 ) in plasma, placental cytosol, and cerebrospinal fluid. While the potential toxicity of small molecules that compete with T4 for binding to TTR should be carefully studied, these small molecules can also serve as anti-ATTR amyloidosis drugs by stabilizing the TTR structure. Here, we demonstrated that rafoxanide, an EU-approved anthelmintic drug for domesticated animals, binds to the T4 -binding site of TTR. An intrinsic fluorescence quenching assay showed that rafoxanide also binds to the thyroid hormone-related proteins, including serum albumin and thyroid hormone receptor ß. Rafoxanide strongly inhibited TTR amyloidogenesis in fibrillization assay, but the binding of rafoxanide to TTR was interfered with in human plasma, probably due to interactions with thyroid hormone-related proteins. Protein crystallography provided clues for the optimization of binding affinity and selectivity. Our findings emphasize the importance of considering rafoxanide as both a possible thyroid-disrupting chemical and a lead compound for the development of new ATTR amyloidosis inhibitors.

Characterization of the molecular interactions between resveratrol derivatives and death-associated protein kinase 1.

Death-associated protein kinase 1 (DAPK1), a Ca2+/calmodulin-regulated serine/threonine kinase, regulates cell apoptosis and autophagy and has been implicated in the pathogenesis of Alzheimer's disease (AD). Targeting DAPK1 may be a promising approach for treating AD. In our previous study, we found that a natural polyphenol, resveratrol (1), is a moderate DAPK1 inhibitor. In the present study, we investigated the interactions between natural and synthetic derivatives of 1 and DAPK1. Binding assays including intrinsic fluorescence quenching, protein thermal shift and isothermal titration calorimetry indicated that oxyresveratrol (3), a hydroxylated derivative, and pinostilbene (5), a methoxylated derivative, bind to DAPK1 with comparable affinity to 1. The enzymatic assay showed that 3 more effectively inhibits the intrinsic ATPase activity of DAPK1 compared with 1. Crystallographic analysis revealed that the binding modes of the methoxylated derivatives were different from those of 1 and 3, resulting in a unique interaction. Our results suggest that 3 may be helpful in treating AD and provide a clue for the development of promising DAPK1 inhibitors.

Hybridization of Wide-Angle X-ray and Neutron Diffraction Techniques in the Crystal Structure Analyses of Synthetic Polymers.

The development in the crystal structure analysis of synthetic polymers using the hybridized combination of wide-angle X-ray and neutron diffraction (WAXD and WAND, respectively) techniques has been reviewed with many case studies performed by the authors. At first, the technical development was reviewed, in which the usage of high-energy synchrotron X-ray source was emphasized for increasing the total number of the observable diffraction peaks, and several examples were introduced. Secondly, the usage of the WAND method was introduced, in which the successful extraction of hydrogen atomic positions was described. The third example is to show the importance for the hybrid combination of these two diffraction methods. The quantitative WAXD data analysis gave the crystal structures of at-poly(vinyl alcohol) (at-PVA) and at-PVA-iodine complex. However, the thus-proposed structure models were found not to reproduce the observed WAND data very much. The reason came from the remarkable difference in the atomic scattering powers of the constituting atomic species between WAXD and WAND phenomena. The introduction of statistical disorder solved this serious problem, which reproduced both of the observed WAXD and WAND data consistently. The more systematic combination of WAXD and WAND methods, or the so-called X-N method, was applied also to the quantitative evaluation of the bonded electron density distribution along the skeletal chains, where the results about polydiacetylene single crystals were presented as the first successful study. Finally, the application of WAND technique in the trace of structural changes induced under the application of external stress or temperature was described. The future perspective is described for the development of structural science of synthetic polymers on the basis of the combined WAXD/WAND techniques.

Neutron crystallography and quantum chemical analysis of bilin reductase PcyA mutants reveal substrate and catalytic residue protonation states.

PcyA, a ferredoxin-dependent bilin pigment reductase, catalyzes the site-specific reduction of the two vinyl groups of biliverdin (BV), producing phycocyanobilin. Previous neutron crystallography detected both the neutral BV and its protonated form (BVH+) in the wildtype (WT) PcyA-BV complex, and a nearby catalytic residue Asp105 was found to have two conformations (protonated and deprotonated). Semiempirical calculations have suggested that the protonation states of BV are reflected in the absorption spectrum of the WT PcyA-BV complex. In the previously determined absorption spectra of the PcyA D105N and I86D mutants, complexed with BV, a peak at 730 nm, observed in the WT, disappeared and increased, respectively. Here, we performed neutron crystallography and quantum chemical analysis of the D105N-BV and I86D-BV complexes to determine the protonation states of BV and the surrounding residues and study the correlation between the absorption spectra and protonation states around BV. Neutron structures elucidated that BV in the D105N mutant is in a neutral state, whereas that in the I86D mutant is dominantly in a protonated state. Glu76 and His88 showed different hydrogen bonding with surrounding residues compared with WT PcyA, further explaining why D105N and I86D have much lower activities for phycocyanobilin synthesis than the WT PcyA. Our quantum mechanics/molecular mechanics calculations of the absorption spectra showed that the spectral change in D105N arises from Glu76 deprotonation, consistent with the neutron structure. Collectively, our findings reveal more mechanistic details of bilin pigment biosynthesis.

Protonation states of hen egg-white lysozyme observed using D/H contrast neutron crystallography.

Hen egg-white lysozyme (HEWL) is an enzymatic protein with two acidic amino acids, Glu35 and Asp52, in its active site. Glu35 acts as a proton donor to the substrate and Asp52 interacts with the positively charged substrate, suggesting different protonation states of these residues. However, neutron crystallographic studies thus far have not provided a consistent picture of the protonation states of these residues. Only one study succeeded in observing the active protonation states of Glu35 and Asp52 in the triclinic crystal system. However, their active states in the most widely studied tetragonal crystal system are still unknown. The application of the D/H contrast technique in neutron crystallography improves the ability to locate exchangeable D/H atoms in proteins. In the present study, D2O and H2O solvent crystals were prepared. Each neutron data set was collected for only five days by combining a time-of-flight diffractometer (iBIX) and the spallation neutron source at the Japan Proton Accelerator Research Complex. The D/H contrast map provided better visualization of the D/H atoms in HEWL than the conventional neutron scattering length density map. The neutron D/H contrast map demonstrated the alternative protonation of the OE1 and OE2 atoms in the carboxyl group of Glu35. This alternative protonation occurs in the absence of a substrate, where high selectivity of the protonation site does not occur. In this case, only the OE1-HE1 bond attacks the substrate in an equilibrium between OE1-HE1 and OE2-HE2, or the H+ ion of the OE2-HE2 bond moves to the OE1 atom just before or after substrate binding to initiate the catalytic reaction. In contrast, the carboxyl group of Asp52 is not protonated. Protonation of the carboxyl group was not observed for other Asp and Glu residues. These results are consistent with results from NMR spectroscopy and explain the protonation states at the active site in the apo form of HEWL.

Re-evaluation of protein neutron crystallography with and without X-ray/neutron joint refinement.

Protein neutron crystallography is a powerful technique to determine the positions of H atoms, providing crucial biochemical information such as the protonation states of catalytic groups and the geometry of hydrogen bonds. Recently, the crystal structure of a bacterial copper amine oxidase was determined by joint refinement using X-ray and neutron diffraction data sets at resolutions of 1.14 and 1.72 Å, respectively [Murakawa et al. (2020 ▸). Proc. Natl Acad. Sci. USA, 117, 10818-10824]. While joint refinement is effective for the determination of the accurate positions of heavy atoms on the basis of the electron density, the structural information on light atoms (hydrogen and deuterium) derived from the neutron diffraction data might be affected by the X-ray data. To unravel the information included in the neutron diffraction data, the structure determination was conducted again using only the neutron diffraction data at 1.72 Šresolution and the results were compared with those obtained in the previous study. Most H and D atoms were identified at essentially the same positions in both the neutron-only and the X-ray/neutron joint refinements. Nevertheless, neutron-only refinement was found to be less effective than joint refinement in providing very accurate heavy-atom coordinates that lead to significant improvement of the neutron scattering length density map, especially for the active-site cofactor. Consequently, it was confirmed that X-ray/neutron joint refinement is crucial for determination of the real chemical structure of the catalytic site of the enzyme.

Revisiting the concept of peptide bond planarity in an iron-sulfur protein by neutron structure analysis.

The planarity of the peptide bond is important for the stability and structure formation of proteins. However, substantial distortion of peptide bonds has been reported in several high-resolution structures and computational analyses. To investigate the peptide bond planarity, including hydrogen atoms, we report a 1.2-Šresolution neutron structure of the oxidized form of high-potential iron-sulfur protein. This high-resolution neutron structure shows that the nucleus positions of the amide protons deviate from the peptide plane and shift toward the acceptors. The planarity of the H─N─C═O plane depends strongly on the pyramidalization of the nitrogen atom. Moreover, the orientation of the amide proton of Cys75 is different in the reduced and oxidized states, possibly because of the electron storage capacity of the iron-sulfur cluster.

Neutron diffraction experiment with the Y116S variant of transthyretin using iBIX at J-PARC: application of a new integration method.

Transthyretin (TTR) is one of more than 30 amyloidogenic proteins, and the amyloid fibrils found in patients afflicted with ATTR amyloidosis are composed of this protein. Wild-type TTR amyloids accumulate in the heart in senile systemic amyloidosis (SSA). ATTR amyloidosis occurs at a much younger age than SSA, and the affected individuals carry a TTR mutant. The naturally occurring amyloidogenic Y116S TTR variant forms more amyloid fibrils than wild-type TTR. Thus, the Y116S mutation reduces the stability of the TTR structure. A neutron diffraction experiment on Y116S TTR was performed to elucidate the mechanism of the changes in structural stability between Y116S variant and wild-type TTR through structural comparison. Large crystals of the Y116S variant were grown under optimal crystallization conditions, and a single 2.4 mm3 crystal was ultimately obtained. This crystal was subjected to time-of-flight (TOF) neutron diffraction using the IBARAKI biological crystal diffractometer (iBIX) at the Japan Proton Accelerator Research Complex, Tokai, Japan (J-PARC). A full data set for neutron structure analysis was obtained in 14 days at an operational accelerator power of 500 kW. A new integration method was developed and showed improved data statistics; the new method was applied to the reduction of the TOF diffraction data from the Y116S variant. Data reduction was completed and the integrated intensities of the Bragg reflections were obtained at 1.9 Šresolution for structure refinement. Moreover, X-ray diffraction data at 1.4 Šresolution were obtained for joint neutron-X-ray refinement.

Towards cryogenic neutron crystallography on the reduced form of [NiFe]-hydrogenase.

A membrane-bound hydrogenase from Desulfovibrio vulgaris Miyazaki F is a metalloenzyme that contains a binuclear Ni-Fe complex in its active site and mainly catalyzes the oxidation of molecular hydrogen to generate a proton gradient in the bacterium. The active-site Ni-Fe complex of the aerobically purified enzyme shows its inactive oxidized form, which can be reactivated through reduction by hydrogen. Here, in order to understand how the oxidized form is reactivated by hydrogen and further to directly evaluate the bridging of a hydride ligand in the reduced form of the Ni-Fe complex, a neutron structure determination was undertaken on single crystals grown in a hydrogen atmosphere. Cryogenic crystallography is being introduced into the neutron diffraction research field as it enables the trapping of short-lived intermediates and the collection of diffraction data to higher resolution. To optimize the cooling of large crystals under anaerobic conditions, the effects on crystal quality were evaluated by X-rays using two typical methods, the use of a cold nitrogen-gas stream and plunge-cooling into liquid nitrogen, and the former was found to be more effective in cooling the crystals uniformly than the latter. Neutron diffraction data for the reactivated enzyme were collected at the Japan Photon Accelerator Research Complex under cryogenic conditions, where the crystal diffracted to a resolution of 2.0 Å. A neutron diffraction experiment on the reduced form was carried out at Oak Ridge National Laboratory under cryogenic conditions and showed diffraction peaks to a resolution of 2.4 Å.

Neutron crystallography of copper amine oxidase reveals keto/enolate interconversion of the quinone cofactor and unusual proton sharing.

Recent advances in neutron crystallographic studies have provided structural bases for quantum behaviors of protons observed in enzymatic reactions. Thus, we resolved the neutron crystal structure of a bacterial copper (Cu) amine oxidase (CAO), which contains a prosthetic Cu ion and a protein-derived redox cofactor, topa quinone (TPQ). We solved hitherto unknown structures of the active site, including a keto/enolate equilibrium of the cofactor with a nonplanar quinone ring, unusual proton sharing between the cofactor and the catalytic base, and metal-induced deprotonation of a histidine residue that coordinates to the Cu. Our findings show a refined active-site structure that gives detailed information on the protonation state of dissociable groups, such as the quinone cofactor, which are critical for catalytic reactions.

Current status and near future plan of neutron protein crystallography at J-PARC.

The IBARAKI Biological Crystal Diffractometer (iBIX) has been available for use at MLF (Material and Life Science Facility) in J-PARC (Japan Proton Accelerator Research Complex) since 2008. The development in state-of-the-art detector systems could enable iBIX to become one of the highest-performance neutron single-crystal diffractometers in the world. Here, together with other various developments, such as data reduction software, crystal growth, and new techniques in measurement coupled analysis, we provided new hydrogen and water structural data of several proteins and macromolecules. Although the proton power at MLF has not yet reached its planned maximum (1MW), a more powerful neutron source will be soon needed for neutron protein crystallography. A future idea is also proposed and discussed in this article.

High-resolution neutron crystallography visualizes an OH-bound resting state of a copper-containing nitrite reductase.

Copper-containing nitrite reductases (CuNIRs) transform nitrite to gaseous nitric oxide, which is a key process in the global nitrogen cycle. The catalytic mechanism has been extensively studied to ultimately achieve rational control of this important geobiochemical reaction. However, accumulated structural biology data show discrepancies with spectroscopic and computational studies; hence, the reaction mechanism is still controversial. In particular, the details of the proton transfer involved in it are largely unknown. This situation arises from the failure of determining positions of hydrogen atoms and protons, which play essential roles at the catalytic site of CuNIRs, even with atomic resolution X-ray crystallography. Here, we determined the 1.50 Šresolution neutron structure of a CuNIR from Geobacillus thermodenitrificans (trimer molecular mass of ∼106 kDa) in its resting state at low pH. Our neutron structure reveals the protonation states of catalytic residues (deprotonated aspartate and protonated histidine), thus providing insights into the catalytic mechanism. We found that a hydroxide ion can exist as a ligand to the catalytic Cu atom in the resting state even at a low pH. This OH-bound Cu site is unexpected from previously given X-ray structures but consistent with a reaction intermediate suggested by computational chemistry. Furthermore, the hydrogen-deuterium exchange ratio in our neutron structure suggests that the intramolecular electron transfer pathway has a hydrogen-bond jump, which is proposed by quantum chemistry. Our study can seamlessly link the structural biology to the computational chemistry of CuNIRs, boosting our understanding of the enzymes at the atomic and electronic levels.

Status of the neutron time-of-flight single-crystal diffraction data-processing software STARGazer.

The STARGazer data-processing software is used for neutron time-of-flight (TOF) single-crystal diffraction data collected using the IBARAKI Biological Crystal Diffractometer (iBIX) at the Japan Proton Accelerator Research Complex (J-PARC). This software creates hkl intensity data from three-dimensional (x, y, TOF) diffraction data. STARGazer is composed of a data-processing component and a data-visualization component. The former is used to calculate the hkl intensity data. The latter displays the three-dimensional diffraction data with searched or predicted peak positions and is used to determine and confirm integration regions. STARGazer has been developed to make it easier to use and to obtain more accurate intensity data. For example, a profile-fitting method for peak integration was developed and the data statistics were improved. STARGazer and its manual, containing installation and data-processing components, have been prepared and provided to iBIX users. This article describes the status of the STARGazer data-processing software and its data-processing algorithms.

Cryoprotectant-free high-pressure cooling and dynamic nuclear polarization for more sensitive detection of hydrogen in neutron protein crystallography.

To improve the sensitivity of hydrogen detection using neutrons, a proton-polarization technique together with a high-pressure cooling method is necessary. The highest pressure (200 MPa) used in the experiment described here enabled relatively large protein crystals to be cooled without any cryoprotectants while retaining the protein structure, and it was confirmed that high-pressure-cooled crystals diffracted to nearly the same resolution as flash-cooled small crystals soaked with cryoprotectants. Dynamic nuclear polarization was used as a proton-polarization technique for protein crystals, and ∼300 mg polycrystalline protein doped with TEMPOL gave a maximum proton polarization of 22.3% at a temperature of 0.5 K in a 2.5 T magnetic field.

Application of profile fitting method to neutron time-of-flight protein single crystal diffraction data collected at the iBIX.

We developed and employed a profile fitting method for the peak integration of neutron time-of-flight diffraction data collected by the IBARAKI Biological Crystal Diffractometer (iBIX) at the Japan Proton Accelerator Research Complex (J-PARC) for protein ribonuclease A and α-thrombin single crystals. In order to determine proper fitting functions, four asymmetric functions were evaluated using strong intensity peaks. A Gaussian convolved with two back-to-back exponentials was selected as the most suitable fitting function, and a profile fitting algorithm for the integration method was developed. The intensity and structure refinement data statistics of the profile fitting method were compared to those of the summation integration method. It was clearly demonstrated that the profile fitting method provides more accurate integrated intensities and model structures than the summation integration method at higher resolution shells. The integration component with the profile fitting method has already been implemented in the iBIX data processing software STARGazer and its user manual has been prepared.

Neutron and X-ray single-crystal diffraction from protein microcrystals via magnetically oriented microcrystal arrays in gels.

Protein microcrystals magnetically aligned in D2O hydrogels were subjected to neutron diffraction measurements, and reflections were observed for the first time to a resolution of 3.4 Šfrom lysozyme microcrystals (∼10 × 10 × 50 µm). This result demonstrated the possibility that magnetically oriented microcrystals consolidated in D2O gels may provide a promising means to obtain single-crystal neutron diffraction from proteins that do not crystallize at the sizes required for neutron diffraction structure determination. In addition, lysozyme microcrystals aligned in H2O hydrogels allowed structure determination at a resolution of 1.76 Šat room temperature by X-ray diffraction. The use of gels has advantages since the microcrystals are measured under hydrated conditions.