dUTP pyrophosphatases from hyperthermophilic eubacterium and archaeon: Structural and functional examinations on the suitability for PCR application.

Deoxyuridine triphosphate pyrophosphatase (DUT) suppresses incorporation of uracil into genomic DNA during replication. Thermostable DUTs from hyperthermophilic archaea such as Thermococcus pacificus enhance PCR amplification by preventing misincorporation of dUTP generated by spontaneous deamination of dCTP. However, it is necessary to elucidate whether DUTs do not cause dNTP imbalances during PCR by unwanted side activity. Moreover, it has been unknown what structural features define the thermostability of those DUTs. Here, DUT from a hyperthermophilic eubacterium, Aquifex aeolicus (Aa-DUT), was characterized together with those from T. pacificus (Tp-DUT). Aa-DUT was as thermostable as Tp-DUT up to at least 95°C. The crystal structures of the two thermostable enzymes were determined, which revealed that the structures of Aa-DUT and Tp-DUT resembled those of monofunctional and bifunctional DUTs, respectively. Generally, bifunctional DUTs harbor the dCTP deaminase activity in addition to the DUT activity. However, not only Aa-DUT but also Tp-DUT showed poor activity towards dCTP, indicating both enzymes are monofunctional. We further examined eight types of parameters related to thermostability of protein structure and found that the thermostability of Aa-DUT and Tp-DUT might be accomplished by increased numbers of ion pairs on the protein surface. Finally, we verified that Aa-DUT promoted PCR amplification with Pfu DNA polymerase to the same extent as Tp-DUT. Collectively, we conclude that both DUTs from hyperthermophiles maintain the enzymatic activity at high temperatures without consuming dCTP due to the lack of the deaminate activity.

Crystal structure of guanosine 5'-monophosphate synthetase from the thermophilic bacterium Thermus thermophilus HB8.

Guanosine 5'-monophosphate (GMP) synthetase (GuaA) catalyzes the last step of GMP synthesis in the purine nucleotide biosynthetic pathway. This enzyme catalyzes a reaction in which xanthine 5'-monophosphate (XMP) is converted to GMP in the presence of Gln and ATP through an adenyl-XMP intermediate. A structure of an XMP-bound form of GuaA from the domain Bacteria has not yet been determined. In this study, the crystal structure of an XMP-bound form of GuaA from the thermophilic bacterium Thermus thermophilus HB8 (TtGuaA) was determined at a resolution of 2.20 Šand that of an apo form of TtGuaA was determined at 2.10 Šresolution. TtGuaA forms a homodimer, and the monomer is composed of three domains, which is a typical structure for GuaA. Disordered regions in the crystal structure were obtained from the AlphaFold2-predicted model structure, and a model with substrates (Gln, XMP and ATP) was constructed for molecular-dynamics (MD) simulations. The structural fluctuations of the TtGuaA dimer as well as the interactions between the active-site residues were analyzed by MD simulations.

Homo-trimeric structure of the ribonuclease for rRNA processing, FAU-1, from Pyrococcus furiosus.

Crystal structure of a ribonuclease for ribosomal RNA processing, FAU-1, from Pyrococcus furiosus was determined with the resolution of 2.57 Å in a homo-trimeric form. The monomer structure consists of two domains: N-terminal and C-terminal domains. C-terminal domain forms trimer and each N-terminal domain locates outside of the trimer core. In the obtained crystal, a dinucleotide, pApUp, was bound to the N-terminal domain, indicating that N-terminal domain has the RNA-binding ability. The affinities to RNA of FAU-1 and a fragment corresponding to the N-terminal domain, FAU-ΔC, were confirmed by polyacrylamide gel electrophoresis and nuclear magnetic resonance (NMR). Interestingly, well-dispersed NMR signals were observed at 318K, indicating that the FAU-ΔC-F18 complex form an ordered structure at higher temperature. As predicted in our previous works, FAU-1 and ribonuclease (RNase) E show a structural similarity in their RNA-binding regions. However, structural similarity between RNase E and FAU-1 could be found in the limited regions of the N-terminal domain. On the other hand, structural similarity between C-terminal domain and some proteins including a phosphatase was found. Thus, it is possible that the catalytic site is located in C-terminal domain.

Catalytic mechanism of the zinc-dependent MutL endonuclease reaction.

DNA mismatch repair endonuclease MutL binds two zinc ions. However, the endonuclease activity of MutL is drastically enhanced by other divalent metals such as manganese, implying that MutL binds another catalytic metal at some site other than the zinc-binding sites. Here, we solved the crystal structure of the endonuclease domain of Aquifex aeolicus MutL in the manganese- or cadmium-bound form, revealing that these metals compete with zinc at the same sites. Mass spectrometry revealed that the MutL yielded 5'-phosphate and 3'-OH products, which is characteristic of the two-metal-ion mechanism. Crystallographic analyses also showed that the position and flexibility of a highly conserved Arg of A. aeolicus MutL altered depending on the presence of zinc/manganese or the specific inhibitor cadmium. Site-directed mutagenesis revealed that the Arg was critical for the catalysis. We propose that zinc ion and its binding sites are physiologically of catalytic importance and that the two-metal-ion mechanism works in the reaction, where the Arg plays a catalytic role. Our results also provide a mechanistic insight into the inhibitory effect of a mutagen/carcinogen, cadmium, on MutL.

Crystal structure of a nucleotide-binding domain of fatty acid kinase FakA from Thermus thermophilus HB8.

Fatty acid kinase is necessary for the incorporation of exogenous fatty acids into membrane phospholipids. Fatty acid kinase consists of two components: a kinase component, FakA, that phosphorylates a fatty acid bound to a fatty acid-binding component, FakB. However, the molecular details underlying the phosphotransfer reaction remain to be resolved. We determined the crystal structure of the N-terminal domain of FakA bound to ADP from Thermus thermophilus HB8. The overall structure of this domain showed that the helical barrel fold is similar to the nucleotide-binding component of dihydroxyacetone kinase. The structure of the nucleotide-binding site revealed the roles of the conserved residues in recognition of ADP and Mg2+, but the N-terminal domain of FakA lacked the ADP-capping loop found in the dihydroxyacetone kinase component. Based on the structural similarity to the two subunits of dihydroxyacetone kinase complex, we constructed a model of the complex of T. thermophilus FakB and the N-terminal domain of FakA. In this model, the invariant Arg residue of FakB occupied a position that was spatially similar to that of the catalytically important Arg residue of dihydroxyacetone kinase, which predicted a composite active site in the Fatty acid kinase complex.

Structures of oxygen dissociation intermediates of 400 kDa V2 hemoglobin provide coarse snapshots of the protein allostery.

Ever since the historic discovery of the cooperative oxygenation of its multiple subunits, hemoglobin (Hb) has been among the most exhaustively studied allosteric proteins. However, the lack of structural information on the intermediates between oxygenated and deoxygenated forms prevents our detailed understanding of the molecular mechanism of its allostery. It has been difficult to prepare crystals of intact oxy-deoxy intermediates and to individually identify the oxygen saturation for each subunit. However, our recent crystallographic studies have demonstrated that giant Hbs from annelids are suitable for overcoming these problems and can provide abundant information on oxy-deoxy intermediate structures. Here, we report the crystal structures of oxy-deoxy intermediates of a 400 kDa Hb (V2Hb) from the annelid Lamellibrachia satsuma, following up on a series of previous studies of similar giant Hbs. Four intermediate structures had average oxygen saturations of 78%, 69%, 55%, and 26%, as determined by the occupancy refinement of the bound oxygen based on ambient temperature factors. The structures demonstrate that the cooperative oxygen dissociation is weaker, large ternary and quaternary changes are induced at a later stage of the oxygen dissociation process, and the ternary and quaternary changes are smaller with local perturbations. Nonetheless, the overall structural transition seemed to proceed in the manner of the MWC two-state model. Our crystallographic snapshots of the allosteric transition of V2Hb provide important experimental evidence for a more detailed understanding of the allostery of Hbs by extension of the Monod-Wyman-Changeux (MWC) model.

Single crystal spectroscopy and multiple structures from one crystal (MSOX) define catalysis in copper nitrite reductases.

Many enzymes utilize redox-coupled centers for performing catalysis where these centers are used to control and regulate the transfer of electrons required for catalysis, whose untimely delivery can lead to a state incapable of binding the substrate, i.e., a dead-end enzyme. Copper nitrite reductases (CuNiRs), which catalyze the reduction of nitrite to nitric oxide (NO), have proven to be a good model system for studying these complex processes including proton-coupled electron transfer (ET) and their orchestration for substrate binding/utilization. Recently, a two-domain CuNiR from a Rhizobia species (Br2DNiR) has been discovered with a substantially lower enzymatic activity where the catalytic type-2 Cu (T2Cu) site is occupied by two water molecules requiring their displacement for the substrate nitrite to bind. Single crystal spectroscopy combined with MSOX (multiple structures from one crystal) for both the as-isolated and nitrite-soaked crystals clearly demonstrate that inter-Cu ET within the coupled T1Cu-T2Cu redox system is heavily gated. Laser-flash photolysis and optical spectroscopy showed rapid ET from photoexcited NADH to the T1Cu center but little or no inter-Cu ET in the absence of nitrite. Furthermore, incomplete reoxidation of the T1Cu site (∼20% electrons transferred) was observed in the presence of nitrite, consistent with a slow formation of NO species in the serial structures of the MSOX movie obtained from the nitrite-soaked crystal, which is likely to be responsible for the lower activity of this CuNiR. Our approach is of direct relevance for studying redox reactions in a wide range of biological systems including metalloproteins that make up at least 30% of all proteins.

In situ crystal data-collection and ligand-screening system at SPring-8.

In situ diffraction data collection using crystallization plates has been utilized for macromolecules to evaluate crystal quality without requiring additional sample treatment such as cryocooling. Although it is difficult to collect complete data sets using this technique due to the mechanical limitation of crystal rotation, recent advances in methods for data collection from multiple crystals have overcome this issue. At SPring-8, an in situ diffraction measurement system was constructed consisting of a goniometer for a plate, an articulated robot and plate storage. Using this system, complete data sets were obtained utilizing the small-wedge measurement method. Combining this system with an acoustic liquid handler to prepare protein-ligand complex crystals by applying fragment compounds to trypsin crystals for in situ soaking, binding was confirmed for seven out of eight compounds. These results show that the system functioned properly to collect complete data for structural analysis and to expand the capability for ligand screening in combination with a liquid dispenser.

Structure of thaumatin under acidic conditions: Structural insight into the conformations in lysine residues responsible for maintaining the sweetness after heat-treatment.

Thaumatin is an intensely sweet-tasting protein. Its sweetness persists when heated under acidic conditions, but disappears when heated at a pH above 7.0. To clarify how the structural features of thaumatin resist insoluble aggregation during heating under acidic conditions, we analysed its crystal structure obtained at pH 4.0, 6.0, and 8.0. Simultaneously, the melting temperature (Tm) at these pH levels was determined using differential scanning fluorimetry. At pH 4.0, the Tm of thaumatin was substantially lower and the overall B-factor value of its structure was higher than those at pH 6.0. Interestingly, the relative B-factor values for most lysine residues decreased as the pH reduced. These results suggest that the overall structure at pH 4.0 becomes flexible but the relative flexibility of some regions is lower than that at pH 6.0. Thus, the reduction in relative flexibility might play an important role in preventing thermal aggregation, thereby maintaining the sweetness.

Structural and functional insights into the mechanism by which MutS2 recognizes a DNA junction.

MutS family proteins are classified into MutS-I and -II lineages: MutS-I recognizes mismatched DNA and initiates mismatch repair, whereas MutS-II recognizes DNA junctions to modulate recombination. MutS-I forms dimeric clamp-like structures enclosing the mismatched DNA, and its composite ATPase sites regulate DNA-binding modes. Meanwhile, the structures of MutS-II have not been determined; accordingly, it remains unknown how MutS-II recognizes DNA junctions and how nucleotides control DNA binding. Here, we solved the ligand-free and ADP-bound crystal structures of bacterial MutS2 belonging to MutS-II. MutS2 also formed a dimeric clamp-like structure with composite ATPase sites. The ADP-bound MutS2 was more flexible compared to the ligand-free form and could be more suitable for DNA entry. The inner hole of the MutS2 clamp was two times larger than that of MutS-I, and site-directed mutagenesis analyses revealed DNA-binding sites at the inner hole. Based on these, a model is proposed that describes how MutS2 recognizes DNA junctions.

Guidelines for de novo phasing using multiple small-wedge data collection. Corrigendum.

A figure in the article by Baba et al. [(2021), J. Synchrotron Rad. 28, 1284-1295] is corrected.

Metagenomic mining and structure-function studies of a hyper-thermostable cellobiohydrolase from hot spring sediment.

Enzymatic breakdown is an attractive cellulose utilisation method with a low environmental load. Its high temperature operation could promote saccharification and lower contamination risk. Here we report a hyper-thermostable cellobiohydrolase (CBH), named HmCel6A and its variant HmCel6A-3SNP that were isolated metagenomically from hot spring sediments and expressed in Escherichia coli. They are classified into glycoside hydrolases family 6 (GH6). HmCel6A-3SNP had three amino acid replacements to HmCel6A (P88S/L230F/F414S) and the optimum temperature at 95 °C, while HmCel6A did it at 75 °C. Crystal structure showed conserved features among GH6, a (ß/α)8-barrel core and catalytic residues, and resembles TfCel6B, a bacterial CBH II of Thermobifida fusca, that had optimum temperature at 60 °C. From structure-function studies, we discuss unique structural features that allow the enzyme to reach its high thermostability level, such as abundance of hydrophobic and charge-charge interactions, characteristic metal bindings and disulphide bonds. Moreover, structure and surface plasmon resonance analysis with oligosaccharides suggested that the contribution of an additional tryptophan located at the tunnel entrance could aid in substrate recognition and thermostability. These results may help to design efficient enzymes and saccharification methods for cellulose working at high temperatures.

Radiation-induced defects in protein crystals observed by X-ray topography.

The characterization of crystal defects induced by irradiation, such as X-rays, charged particles and neutrons, is important for understanding radiation damage and the associated generation of defects. Radiation damage to protein crystals has been measured using various methods. Until now, these methods have focused on decreased diffraction intensity, volume expansion of unit cells and specific damage to side chains. Here, the direct observation of specific crystal defects, such as dislocations, induced by X-ray irradiation of protein crystals at room temperature is reported. Dislocations are induced even by low absorbed doses of X-ray irradiation. This study revealed that for the same total absorbed dose, the formation of defects appears to critically depend on the dose rate. The relationship between dislocation energy and dose energy was analyzed based on dislocation theory associated with elasticity theory for crystalline materials. This demonstration of the crystal defects induced by X-ray irradiation could help to understand the underlying mechanisms of X-ray-induced radiation damage.

Coarse snapshots of oxygen-dissociation intermediates of a giant hemoglobin elucidated by determining the oxygen saturation in individual subunits in the crystalline state.

Cooperative oxygen binding of hemoglobin (Hb) has been studied for over half a century as a representative example of the allostericity of proteins. The most important problem remaining to be solved is the lack of structural information on the intermediates between the oxygenated and deoxygenated forms. In order to characterize the intermediate structures, it is necessary to obtain intermediate-state crystals, determine their oxygen saturations and then determine the oxygen saturations of each of their constituent subunits, all of which are challenging issues even now. Here, intermediate forms of the 400 kDa giant Hb from the tubeworm Oligobrachia mashikoi are reported. To overcome the above problems without any artificial modifications to the protein or prosthetic groups, intermediate crystals of the giant Hb were prepared from fully oxygenated crystals by a soaking method. The oxygen saturation of the crystals was measured by in situ observation with a microspectrophotometer using thin plate crystals processed by an ultraviolet laser to avoid saturation of absorption. The oxygen saturation of each subunit was determined by occupancy refinement of the bound oxygen based on ambient temperature factors. The obtained structures reveal the detailed relationship between the structural transition and oxygen dissociation. The dimer subassembly of the giant Hb shows strong correlation with the local structural changes at the heme pockets. Although some local ternary-structural changes occur in the early stages of the structural transition, the associated global ternary-structural and quaternary-structural changes might arise at about 50% oxygen saturation. The models based on coarse snapshots of the allosteric transition support the conventional two-state model of Hbs and provide the missing pieces of the intermediate structures that are required for full understanding of the allosteric nature of Hbs in detail.

Guidelines for de novo phasing using multiple small-wedge data collection.

Intense micro-focus X-ray beamlines available at synchrotron facilities have achieved high-quality data collection even from the microcrystals of membrane proteins. The automatic data collection system developed at SPring-8, named ZOO, has contributed to many structure determinations of membrane proteins using small-wedge synchrotron crystallography (SWSX) datasets. The `small-wedge' (5-20°) datasets are collected from multiple crystals and then merged to obtain the final structure factors. To our knowledge, no systematic investigation on the dose dependence of data accuracy has so far been reported for SWSX, which is between `serial crystallography' and `rotation crystallography'. Thus, herein, we investigated the optimal dose conditions for experimental phasing with SWSX. Phase determination using anomalous scattering signals was found to be more difficult at higher doses. Furthermore, merging more homogeneous datasets grouped by hierarchical clustering with controlled doses mildly reduced the negative factors in data collection, such as `lack of signal' and `radiation damage'. In turn, as more datasets were merged, more probable phases could be obtained across a wider range of doses. Therefore, our findings show that it is essential to choose a lower dose than 10 MGy for de novo structure determination by SWSX. In particular, data collection using a dose of 5 MGy proved to be optimal in balancing the amount of signal available while reducing the amount of damage as much as possible.

Evaluation of the data-collection strategy for room-temperature micro-crystallography studied by serial synchrotron rotation crystallography combined with the humid air and glue-coating method.

Synchrotron serial crystallography (SSX) is an emerging data-collection method for micro-crystallography on synchrotron macromolecular (MX) crystallography beamlines. At SPring-8, the feasibility of the fixed-target approach was examined by collecting data using a 2D raster scan combined with goniometer rotation. Results at cryogenic temperatures demonstrated that rotation is effective for efficient data collection in SSX and the method was named serial synchrotron rotation crystallography (SS-ROX). To use this method for room-temperature (RT) data collection, a humid air and glue-coating (HAG) method was developed in which data were collected from polyvinyl alcohol-coated microcrystals fixed on a loop under humidity-controlled air. The performance and the RT data-collection strategy for micro-crystallography were evaluated using microcrystals of lysozyme. Although a change in unit-cell dimensions of up to 1% was observed during data collection, the impact on data quality was marginal. A comparison of data obtained at various absorbed doses revealed that absorbed doses of up to 210 kGy were tolerable in both global and local damage. Although this limits the number of photons deposited on each crystal, increasing the number of merged images improved the resolution. On the basis of these results, an equation was proposed that relates the achievable resolution to the total photon flux used to obtain a data set.

Computer-controlled liquid-nitrogen drizzling device for removing frost from cryopreserved crystals.

Cryocrystallography is a technique that is used more often than room-temperature data collection in macromolecular crystallography. One of its advantages is the significant reduction in radiation damage, which is especially useful in synchrotron experiments. Another advantage is that cryopreservation provides simple storage of crystals and easy transportation to a synchrotron. However, this technique sometimes results in the undesirable adhesion of frost to mounted crystals. The frost produces noisy diffraction images and reduces the optical visibility of crystals, which is crucial for aligning the crystal position with the incident X-ray position. To resolve these issues, a computer-controlled device has been developed that drizzles liquid nitrogen over a crystal to remove frost. It was confirmed that the device works properly, reduces noise from ice rings in diffraction images and enables the centering of crystals with low visibility owing to frost adhesion.

A Lynch syndrome-associated mutation at a Bergerat ATP-binding fold destabilizes the structure of the DNA mismatch repair endonuclease MutL.

In humans, mutations in genes encoding homologs of the DNA mismatch repair endonuclease MutL cause a hereditary cancer that is known as Lynch syndrome. Here, we determined the crystal structures of the N-terminal domain (NTD) of MutL from the thermophilic eubacterium Aquifex aeolicus (aqMutL) complexed with ATP analogs at 1.69-1.73 Å. The structures revealed significant structural similarities to those of a human MutL homolog, postmeiotic segregation increased 2 (PMS2). We introduced five Lynch syndrome-associated mutations clinically found in human PMS2 into the aqMutL NTD and investigated the protein stability, ATPase activity, and DNA-binding ability of these protein variants. Among the mutations studied, the most unexpected results were obtained for the residue Ser34. Ser34 (Ser46 in PMS2) is located at a previously identified Bergerat ATP-binding fold. We found that the S34I aqMutL NTD retains ATPase and DNA-binding activities. Interestingly, CD spectrometry and trypsin-limited proteolysis indicated the disruption of a secondary structure element of the S34I NTD, destabilizing the overall structure of the aqMutL NTD. In agreement with this, the recombinant human PMS2 S46I NTD was easily digested in the host Escherichia coli cells. Moreover, other mutations resulted in reduced DNA-binding or ATPase activity. In summary, using the thermostable aqMutL protein as a model molecule, we have experimentally determined the effects of the mutations on MutL endonuclease; we discuss the pathological effects of the corresponding mutations in human PMS2.

The high-resolution crystal structure of lobster hemocyanin shows its enzymatic capability as a phenoloxidase.

Hemocyanin (Hc) and phenoloxidase (PO) are members of the type 3 copper protein family. Although arthropod Hc and PO exhibit similar three-dimensional structures of the copper-containing active site, Hc functions as an oxygen transport protein, showing minimal or no phenoloxidase activity. Here, we present the crystal structure of the oxy form of Hc from Panulirus japonicus (PjHc) at 1.58 Å resolution. The structure of the di-copper active site of PjHc was found to be almost identical to that of PO. Although conserved amino acids and the water molecule crucial for the enzymatic activity were observed in PjHc at almost the same positions as those in PO, PjHc showed no enzymatic activity under our experimental conditions. One striking difference between PjHc and arthropod PO was the presence of a "blocker residue" near the binuclear copper site of PjHc. This blocker residue comprised a phenylalanine residue tightly stacked with an imidazole ring of a CuA coordinated histidine and hindered substrates from accessing the active site. Our results suggest that the blocker residue is also a determining factor of the catalytic activity of type 3 copper proteins.

X-ray dose-dependent structural changes of the [2Fe-2S] ferredoxin from Chlamydomonas reinhardtii.

Plant-type ferredoxin (Fd) is an electron transfer protein in chloroplast. Redox-dependent structural change of Fd controls its association with and dissociation from Fd-dependent enzymes. Among many X-ray structures of oxidized Fd have been reported so far, very likely a given number of them was partially reduced by strong X-ray. To understand the precise structural change between reduced and oxidized Fd, it is important to know whether the crystals of oxidized Fd may or may not be reduced during the X-ray experiment. We prepared the thin plate-shaped Fd crystals from Chlamydomonas reinhardtii and monitored its absorption spectra during experiment. Absorption spectra of oxidized Fd crystals were clearly changed to that of reduced form in an X-ray dose-dependent manner. In another independent experiment, the X-ray diffraction images obtained from different parts of one single crystal were sorted and merged to form two datasets with low and high X-ray doses. An Fo-Fo map calculated from the two datasets showed that X-ray reduction causes a small displacement of the iron atoms in the [2Fe-2S] cluster. Both our spectroscopic and crystallographic studies confirm X-ray dose-dependent reduction of Fd, and suggest a structural basis for its initial reduction step especially in the core of the cluster.