Heat shock protein 90 (Hsp90) is a molecular chaperone that facilitates the folding and maturation of client proteins and is implicated in diseases such as cancer. Of its three domains-N-terminal, middle, and C-terminal-the N-terminal domain (NTD) plays a role in ATP hydrolysis, which is required for the chaperone function of Hsp90 in vivo. Precise information about ATP (and ADP) recognition is useful for designing drugs that target the ATP-binding site. X-ray crystal structures of human Hsp90α-NTD have revealed hydrogen bonds around the ADP-binding site, but the exact hydrogen-bond patterns are unclear, mainly because their structures lack the hydrogen atoms essential for identifying donor and acceptor pairs. Here, we performed neutron structural analyses of human Hsp90α-NTD in both its apo and ADP-bound forms. These structures clarify the hydrogen bonds, including the orientations of water molecules. The neutron structures show that ADP and magnesium binding do not perturb the apo state hydrogen-bond network. The orientation of a water molecule and the position of the Met98 side chain indicate that lone pair-π and CH-π interactions stabilize the binding of the adenine ring. A Hsp90-specific intramolecular hydrogen bond between the ribose moiety and the phosphate implies its contribution to the ATP hydrolysis catalyzed by Hsp90. The precise view of the ADP-binding site indicates that the unperturbed hydration structure plays an important role in ADP recognition.

RhiM and RhiC are ATP-dependent l-amino acid ligases involved in the biosynthesis of the antifungal peptide rhizocticin. RhiM catalyzes the ligation of l-arginine with a phosphonic amino acid, and RhiC subsequently ligates the RhiM product with hydrophobic amino acids. Although biochemical studies have clarified the enzymatic functions of these ligases, the molecular basis of the catalytic mechanisms and substrate selectivity is unknown because there is no structural information for nucleotide- and substrate-bound states or direct structural evidence for the proposed acyl phosphate intermediate. In this study, we performed X-ray crystallographic and biochemical analyses of RhiM and RhiC. Crystal structures were determined for the nucleotide-bound forms of the enzymes as well as the substrate- and intermediate-analog-bound forms of RhiC. The structure of RhiC bound to an intermediate analog provides structural evidence supporting a mechanism where catalysis proceeding via an acyl phosphate intermediate. Structural comparisons of the previously reported apo form of RhiM and the multiple reaction-state structures determined here indicate that closure of three catalytic loops is essential for forming the substrate binding pocket and the correct positioning of the substrate and intermediates in the active site. The hydrophilic and hydrophobic pockets formed by these loops are optimally configured to accommodate the RhiM and RhiC substrates. Mutational analyses confirm that residues involved in substrate binding and Mg2+ coordination are critical for the ligase activity. These findings show that conformational rearrangements of the catalytic loops govern catalytic reaction and ligand selectivity in these enzymes.

Many structural studies have been reported for ferredoxin:NADP+ reductase family members, but an experimental validation of the catalytic hydride and proton transfer steps through a direct detection of the involved hydrogen atoms has not been achieved so far. Here, we determined high-resolution X-ray and neutron crystal structures of NADH-cytochrome b5 reductase, which acts as an electron supplier for various metabolic processes and mediates hydride and proton transfer reactions via its FAD and NADH cofactors. The X-ray structures identify the FADH--NAD+ and FAD-NADH complexes based on the electron densities of the hydrogen atoms bound to the cofactors. The neutron structures determined at different pD-values show a difference in the protonation state of the histidine residue in the hydrogen-bond network from FAD to the protein surface. The observation of the hydrogen atoms reveals the structural basis for the hydride and proton transfer reactions catalyzed by NADH-cytochrome b5 reductase.

About 70 years ago, the crystal structure of the nylon 6 alpha form was proposed by Bunn et al. (Bunn's model) on the basis of wide-angle X-ray diffraction (WAXD) data analysis. The nearly extended planar-zigzag chains with alternating upward and downward orientations are regularly connected by intermolecular hydrogen bonds to form a sheet plane. These sheets are stacked regularly under the space group symmetry of P21. The quantitative analyses of WAXD and WAND data, which were measured for the first time in the present study, revealed the following. (i) Bunn's model can be constructed using the space group P21/n of higher symmetry (this revision is important in the structure-property discussion), (ii) but their regular model cannot reproduce both WAXD and WAND data without any issues. Moreover, (iii) the introduction of a statistically disordered up/down chain packing concept can reproduce these observed diffraction data consistently. As one local structure, the aggregation of domains has been proposed, where the domain itself consists of regular chain packing, but these domains are randomly aggregated together with the +/- a/2 shift and a small c-axial shift to form a crystalline lamella. In this way, the crystal structure of the nylon-6 alpha form has been conclusively established.

Cytochrome P450 (CYP) enzymes bind a heme group that acts as a catalytic center. Inhibition mechanisms in CYP enzymes have been studied extensively by biochemical and structural analyses. Noncompetitive inhibitors are generally believed to bind to allosteric sites remote from the active site to form enzyme-substrate-inhibitor (ESI) complexes. Docking simulations predict the binding sites of noncompetitive inhibitors to CYP enzymes, but to date, there has been no experimental structural verification of ESI complexes formed by CYP enzymes. We performed biochemical and structural analyses of CYP105A1 using the imidazole-containing inhibitors ketoconazole, lanoconazole, and miconazole. Enzyme inhibition analyses demonstrated that ketoconazole and miconazole act as competitive inhibitors, whereas lanoconazole acts as a noncompetitive inhibitor of CYP105A1. The obtained X-ray structures of enzyme-inhibitor (EI) complexes showed that lanoconazole can bind in various orientations to the heme iron compared with ketoconazole and miconazole. We also determined the X-ray structure of an ESI complex comprising CYP105A1, diclofenac, and lanoconazole. This structure shows that lanoconazole binds to the heme iron and that diclofenac closely interacts with the bound lanoconazole, but it is positioned distant from the heme group. Quantum mechanical calculations indicate that Cl-π and electrostatic interactions between diclofenac and lanoconazole, and electrostatic interactions between diclofenac and positively charged arginine residues, stabilize the formation of the ESI complex. Based on these results, we propose a mechanism for cooperative inhibition between a substrate and an apparent noncompetitive inhibitor.

Human MTH1, a Nudix enzyme, hydrolyzes several oxidized nucleotides such as 8-oxo-dGTP and 2-oxo-dATP, owing to its broad substrate specificity. MTH1 has also attracted attention as an anticancer target, and its substrate recognition is of biological and medical interest. Previous studies have suggested that MTH1 exhibits broad substrate specificity by changing the protonation states of Asp119 and Asp120 with high pKa. However, the recognition mechanism remains unclear, owing to the difficulty in directly observing hydrogen atoms. Furthermore, a recent time-resolved X-ray study has proposed that Nudix hydrolases catalyze reactions through a new three-metal-ion mechanism. To understand the substrate recognition and catalytic mechanisms of human MTH1, we performed neutron and time-resolved X-ray crystallography. The neutron structures of MTH1 complexed with 8-oxo-dGTP and 2-oxo-dATP revealed the protonation states of the active-site residues, substrates, and water molecules, crucial for substrate binding and catalysis, providing direct experimental evidence that changes in the protonation states of Asp119 and Asp120 enable broad substrate recognition of MTH1. Time-resolved X-ray crystallography was used to visualize the entire reaction process through Mn2+ ion. The combination of neutron and time-resolved X-ray crystallography led to the proposal of a reaction mechanism for MTH1 via three metal-binding sites, including the conformational dynamics of a loop region, nucleophilic substitution, and a potential deprotonation pathway. Overall, the mechanism involving three metal-binding sites may be a general feature in the catalysis of Nudix hydrolases.

Ionizing radiation induces various types of DNA damage, and the reparability and lethal effects of DNA damage differ depending on its spatial density. Elucidating the structure of radiation-induced clustered DNA damage and its repair processes will enhance our understanding of the lethal impact of ionizing radiation and advance progress toward precise therapeutics. Previously, we developed a method to directly visualize DNA damage using atomic force microscopy (AFM) and classified clustered DNA damage into simple base damage clusters (BDCs), complex BDCs and complex double-strand breaks (DSBs). This study investigated the repair of each type of damage in DNA-repair-deficient human TK6 cells and elucidated the association between each type of clustered DNA damage and the pathway responsible for its repair postirradiation with low linear energy transfer (LET) radiation (X-rays) and high-LET radiation (Fe-ion beams) in cells. We found that base excision repair and, surprisingly, nucleotide excision repair restored simple and complex BDCs. In addition, the number of complex DSBs in wild-type cells increases 1 h postirradiation, which was most likely caused by BDC cleavage initiated with DNA glycosylases. Furthermore, complex DSBs, which are likely associated with lethality, are repaired by homologous recombination with little contribution from nonhomologous-end joining.

Hydrogen (H) atoms account for about half the atoms in biomacromolecules and are essential for their biochemical properties such as enzymatic functions. Obtaining precise enzyme structures that include all the H atoms allows a deeper understanding of their structure-function relationships. Copper-containing nitrite reductases (CuNIRs) catalyze transformation of nitrite to nitric oxide, which has impacts on geochemical, agricultural, and medical health fields. Despite intense research efforts, the dynamics of H atoms during the enzymatic reaction of CuNIRs are unknown and hence the catalytic mechanism remains unclear. We performed neutron crystallography to shoot a single H-atom resolution picture of a CuNIR in complex with nitrite. We found that nitrite binds on the catalytic Cu center as nitrite (NO2-) and not as protonated HNO2. Our X-ray data and quantum chemical calculation show that NO2- is in an electron-localized state that can facilitate N-O bond cleavage after receiving an electron. The catalytic residues, AspCAT and HisCAT, are deprotonated and protonated, respectively, suggesting that HisCAT is the point of departure of the proton transfer sequence. Quantum chemical calculations show that the neutron structure is consistent with the Cu(II) state and that the highly polarized state of the catalytic site is stabilized by the permittivity of solvent molecules filling a water channel. Subatomic resolution X-ray structures of the AspCAT-to-Asn mutants, which mimic the protonated state of AspCAT, were also determined to investigate the involvement of protonated AspCAT in the reaction. Our crystallographic data and quantum chemical calculations reveal in detail the first step of the CuNIR reaction.

Nylon-6 exhibits several forms of crystal modification. The α form converts to the iodine complex when it is immersed in a highly concentrated KI/I2 solution. After deiodization in a hypo (sodium thiosulfate) solution, the iodine complex changes to the γ form. The phase transition mechanism from the α to the γ form through the iodine complex has remained a challenging issue because of the lack of established crystal structures. As previously reported (Polymer Journal, 2025), a quantitative analysis of 2D wide-angle X-ray and neutron diffraction data necessitated a revision of the crystal structure of the α form by incorporating up/down chain packing disorder. This paper reports that a similar up/down chain disorder is also present in the crystal lattices of the γ form and the iodine complexes. In both the α and γ forms, the crystal lattice is composed of stacked hydrogen-bonded sheet planes. The aforementioned up/down chain packing disorder can be expressed in terms of the stacking disorder of sheets or the disordered slippages of sheets along the a axis. Thus, the notion of stacking disorder of sheet planes allows for a systematic and logical interpretation of the transition behaviors among these crystalline forms of nylon-6. Analysis of wide-angle X-ray and neutron diffraction data has shown that the crystal structures of nylon-6 in both α and γ forms, as well as the iodine complex, are composed of statistically disordered arrangements of molecular chains. This composition can alternatively be described as the disordered slippages of sheet planes. The notion of stacking disorder within these sheets enables a logical interpretation of the transition mechanism among the three crystalline phases.

Metalloproteins play fundamental roles in organisms and are utilized as starting points for the directed evolution of artificial enzymes. Knowing the strategies of metalloproteins, by which they exquisitely tune their activities, will not only lead to an understanding of biochemical phenomena but also contribute to various applications. The blue copper protein (BCP) has been a renowned model system to understand the biology, chemistry, and physics of metalloproteins. Pseudoazurin (Paz), a blue copper protein, mediates electron transfer in the bacterial anaerobic respiratory chain. Its redox potential is finely tuned by hydrogen (H) bond networks; however, difficulty in visualizing H atom positions in the protein hinders the detailed understanding of the protein's structure-function relationship. We here used neutron and sub-angstrom resolution X-ray crystallography to directly observe H atoms in Paz. The 0.86-angstrom-resolution X-ray structure shows that the peptide bond between Pro80 and the His81 Cu ligand deviates from the ideal planar structure. The 1.9-angstrom-resolution neutron structure confirms a long-overlooked H bond formed by the amide of His81 and the S atom of another Cu ligand Cys78. Quantum mechanics/molecular mechanics calculations show that this H bond increases the redox potential of the Cu site and explains the experimental results well. Our study demonstrates the potential of neutron and sub-angstro''m resolution X-ray crystallography to understand the chemistry of metalloproteins at atomic and quantum levels.

NCYM is a cis-antisense gene of MYCN oncogene and encodes an oncogenic protein that stabilizes MYCN via inhibition of GSK3b. High NCYM expression levels are associated with poor clinical outcomes in human neuroblastomas, and NCYM overexpression promotes distant metastasis in animal models of neuroblastoma. Using vacuum-ultraviolet circular dichroism and small-angle X-ray scattering, we previously showed that NCYM has high flexibility with partially folded structures; however, further structural characterization is required for the design of anti-cancer agents targeting NCYM. Here we report the 1H, 15N and 13C nuclear magnetic resonance assignments of NCYM. Secondary structure prediction using Secondary Chemical Shifts and TALOS-N analysis demonstrates that the structure of NCYM is essentially disordered, even though residues in the central region of the peptide clearly present a propensity to adopt a dynamic helical structure. This preliminary study provides foundations for further analysis of interaction between NCYM and potential partners.

NCYM is a cis-antisense gene of MYCN oncogene and encodes an oncogenic protein that stabilizes MYCN via inhibition of GSK3b. High NCYM expression levels are associated with poor clinical outcomes in human neuroblastomas, and NCYM overexpression promotes distant metastasis in animal models of neuroblastoma. Using vacuum-ultraviolet circular dichroism and small-angle X-ray scattering, we previously showed that NCYM has high flexibility with partially folded structures; however, further structural characterization is required for the design of anti-cancer agents targeting NCYM. Here we report the 1 H, 15 N and 13 C nuclear magnetic resonance assignments of NCYM. Secondary structure prediction using Secondary Chemical Shifts and TALOS-N analysis demonstrates that the structure of NCYM is essentially disordered, even though residues in the central region of the peptide clearly present a propensity to adopt a dynamic helical structure. This preliminary study provides foundations for further analysis of interaction between NCYM and potential partners.

To improve the specific activity at low temperatures of Ef-EG2 and to maintain thermostability, five mutant enzymes (K273R, N372D, Q387E, N402D, D43R) were produced. The two mutant enzymes (K273R, N402D) lost the cellulase activity. The specific activities of mutant N372D, Q387E, and D43R enzymes were 2.5-fold higher than that of WT enzyme over various temperatures. The denaturation temperatures (Tm) of WT and N372D, Q387E, and D43R enzymes were 55.1 degrees C, 51.5 degrees C, 51.2 degrees C, and 55.6 degrees C. D43R showed almost the same ther-mostability as the WT enzyme. The three-dimensional structure of D43R was not significantly different from that of the WT enzyme, but the D43R enzyme lost the ability to bind sodium ions. D43R was introduced the additional electrostatic interaction with Asp55.

Biological properties of protein molecules depend on their interaction with other molecules, and enzymes are no exception. Enzyme activities are controlled by their interaction with other molecules in living cells. Enzyme activation and their catalytic properties in the presence of different types of polymers have been studied in vitro, although these studies are restricted to only a few enzymes. In this study, we show that addition of poly-l-lysine (PLL) can increase the enzymatic activity of multiple oxidoreductases through formation of enzyme assemblies. Oxidoreductases with an overall negative charge, such as l-lactate oxidase, d-lactate dehydrogenase, pyruvate oxidase, and acetaldehyde dehydrogenase, each formed assemblies with the positively charged PLL via electrostatic interactions. The enzyme activities of these oxidoreductases in the enzyme assemblies were several-folds higher than those of the enzyme in their natural dispersed state. In the presence of PLL, the turnover number (kcat) improved for all enzymes, whereas the decrease in Michaelis constant (KM) was enzyme dependent. This type of enzyme function regulation through the formation of assemblies via simple addition of polymers has potential for diverse applications, including various industrial and research purposes.

NCYM, a Homininae-specific oncoprotein, is the first de novo gene product experimentally shown to have oncogenic functions. NCYM stabilizes MYCN and β-catenin via direct binding and inhibition of GSK3β and promotes cancer progression in various tumors. Thus, the identification of compounds that binds to NCYM and structural characterization of the complex of such compounds with NCYM are required to deepen our understanding of the molecular mechanism of NCYM function and eventually to develop anticancer drugs against NCYM. In this study, the DNA aptamer that specifically binds to NCYM and enhances interaction between NCYM and GSK3β were identified for the first time using systematic evolution of ligands by exponential enrichment (SELEX). The structural properties of the complex of the aptamer and NCYM were investigated using atomic force microscopy (AFM) in combination with truncation and mutation of DNA sequence, pointing to the regions on the aptamer required for NCYM binding. Further analysis was carried out by small-angle X-ray scattering (SAXS). Structural modeling based on SAXS data revealed that when isolated, NCYM shows high flexibility, though not as a random coil, while the DNA aptamer exists as a dimer in solution. In the complex state, models in which NCYM was bound to a region close to an edge of the aptamer reproduced the SAXS data. Therefore, using a combination of SELEX, AFM, and SAXS, the present study revealed the structural properties of NCYM in its functionally active form, thus providing useful information for the possible future design of novel anti-cancer drugs targeting NCYM.