1H, 13C and 15N backbone and side-chain resonance assignments of the human oncogenic protein NCYM.

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.

Overlooked Hydrogen Bond in a Blue Copper Protein Uncovered by Neutron and Sub-Ångström Resolution X-ray Crystallography.

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-ångström resolution X-ray crystallography to directly observe H atoms in Paz. The 0.86-Å-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-Å-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-ångström resolution X-ray crystallography to understand the chemistry of metalloproteins at atomic and quantum levels.

Structural characterization of human de novo protein NCYM and its complex with a newly identified DNA aptamer using atomic force microscopy and small-angle X-ray scattering.

NCYM, a Homininae-specific oncoprotein, is the first de novo gene product experimentally shown to have oncogenic functions. NCYM stabilizes MYCN and ß-catenin via direct binding and inhibition of GSK3ß 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 GSK3ß 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.

Description of peptide bond planarity from high-resolution neutron crystallography.

Neutron crystallography is a highly effective method for visualizing hydrogen atoms in proteins. In our recent study, we successfully determined the high-resolution (1.2 Å) neutron structure of high-potential iron-sulfur protein, refining the coordinates of some amide protons without any geometric restraints. Interestingly, we observed that amide protons are deviated from the peptide plane due to electrostatic interactions. Moreover, the difference in the position of the amide proton of Cys75 between reduced and oxidized states is possibly attributed to the electron storage capacity of the iron-sulfur cluster. Additionally, we have discussed about the rigidity of the iron-sulfur cluster based on the results of the hydrogen-deuterium exchange. Our research underscores the significance of neutron crystallography in protein structure elucidation, enriching our understanding of protein functions at an atomic resolution.

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.

Activation of oxidoreductases by the formation of enzyme assembly.

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.

Creation of Cross-Linked Crystals With Intermolecular Disulfide Bonds Connecting Symmetry-Related Molecules Allows Retention of Tertiary Structure in Different Solvent Conditions.

Protein crystals are generally fragile and sensitive to subtle changes such as pH, ionic strength, and/or temperature in their crystallization mother liquor. Here, using T4 phage lysozyme as a model protein, the three-dimensional rigidification of protein crystals was conducted by introducing disulfide cross-links between neighboring molecules in the crystal. The effect of cross-linking on the stability of the crystals was evaluated by microscopic observation and X-ray diffraction. When soaking the obtained cross-linked crystals into a precipitant-free solution, the crystals held their shape without dissolution and diffracted to approximately 1.1 Å resolution, comparable to that of the non-cross-linked crystals. Such cross-linked crystals maintained their diffraction even when immersed in other solutions with pH values from 4 to 10, indicating that the disulfide cross-linking made the packing contacts enforced and resulted in some mechanical strength in response to changes in the preservation conditions. Furthermore, the cross-linked crystals gained stability to permit soaking into solutions containing high concentrations of organic solvents. The results suggest the possibility of obtaining protein crystals for effective drug screening by introducing appropriate cross-linked disulfide bonds.

Current status of neutron crystallography in structural biology.

Hydrogen atoms and hydration water molecules in proteins are essential for many biochemical processes, especially enzyme catalysis. Neutron crystallography enables direct observation of hydrogen atoms, and reveals molecular recognition through hydrogen bonding and catalytic reactions involving proton-coupled electron transfer. The use of neutron crystallography is still limited for proteins, but its popularity is increasing owing to an increase in the number of diffractometers for structural biology at neutron facilities and advances in sample preparation. According to the characteristics of the neutrons, monochromatic or quasi-Laue methods and the time-of-flight method are used in nuclear reactors and pulsed spallation sources, respectively, to collect diffraction data. Growing large crystals is an inevitable problem in neutron crystallography for structural biology, but sample deuteration, especially protein perdeuteration, is effective in reducing background levels, which shortens data collection time and decreases the crystal size required. This review also introduces our recent neutron structure analyses of copper amine oxidase and copper-containing nitrite reductase. The neutron structure of copper amine oxidase gives detailed information on the protonation state of dissociable groups, such as the quinone cofactor, which are critical for catalytic reactions. Electron transfer via a hydrogen-bond jump and a hydroxide ion ligation in copper-containing nitrite reductase are clarified, and these observations are consistent with the results from the quantum chemical calculations. This review article is an extended version of the Japanese article, Elucidation of Enzymatic Reaction Mechanism by Neutron Crystallography, published in SEIBUTSU-BUTSURI Vol. 61, p.216-222 (2021).

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.

Formation of clustered DNA damage in vivo upon irradiation with ionizing radiation: Visualization and analysis with atomic force microscopy.

SignificanceDNA damage causes loss of or alterations in genetic information, resulting in cell death or mutations. Ionizing radiations produce local, multiple DNA damage sites called clustered DNA damage. In this study, a complete protocol was established to analyze the damage complexity of clustered DNA damage, wherein damage-containing genomic DNA fragments were selectively concentrated via pulldown, and clustered DNA damage was visualized by atomic force microscopy. It was found that X-rays and Fe ion beams caused clustered DNA damage. Fe ion beams also produced clustered DNA damage with high complexity. Fe ion beam-induced complex DNA double-strand breaks (DSBs) containing one or more base lesion(s) near the DSB end were refractory to repair, implying their lethal effects.

Hydration and its Hydrogen Bonding State on a Protein Surface in the Crystalline State as Revealed by Molecular Dynamics Simulation.

Protein hydration is crucial for the stability and molecular recognition of a protein. Water molecules form a hydration water network on a protein surface via hydrogen bonds. This study examined the hydration structure and hydrogen bonding state of a protein, staphylococcal nuclease, at various hydration levels in its crystalline state by all-atom molecular dynamics (MD) simulation. Hydrophilic residues were more hydrated than hydrophobic residues. As the water content increases, both types of residues were uniformly more hydrated. The number of hydrogen bonds per single water asymptotically approaches 4, the same as bulk water. The distances and angles of hydrogen bonds in hydration water in the protein crystal were almost the same as those in the tetrahedral structure of bulk water regardless of the hydration level. The hydrogen bond structure of hydration water observed by MD simulations of the protein crystalline state was compared to the Hydrogen and Hydration Database for Biomolecule from experimental protein crystals.

Secondary Structure of Human De Novo Evolved Gene Product NCYM Analyzed by Vacuum-Ultraviolet Circular Dichroism.

NCYM, a cis-antisense gene of MYCN, encodes a Homininae-specific protein that promotes the aggressiveness of human tumors. Newly evolved genes from non-genic regions are known as de novo genes, and NCYM was the first de novo gene whose oncogenic functions were validated in vivo. Targeting NCYM using drugs is a potential strategy for cancer therapy; however, the NCYM structure must be determined before drug design. In this study, we employed vacuum-ultraviolet circular dichroism to evaluate the secondary structure of NCYM. The SUMO-tagged NCYM and the isolated SUMO tag in both hydrogenated and perdeuterated forms were synthesized and purified in a cell-free in vitro system, and vacuum-ultraviolet circular dichroism spectra were measured. Significant differences between the tagged NCYM and the isolated tag were evident in the wavelength range of 190-240 nm. The circular dichroism spectral data combined with a neural network system enabled to predict the secondary structure of NCYM at the amino acid level. The 129-residue tag consists of α-helices (approximately 14%) and ß-strands (approximately 29%), which corresponded to the values calculated from the atomic structure of the tag. The 238-residue tagged NCYM contained approximately 17% α-helices and 27% ß-strands. The location of the secondary structure predicted using the neural network revealed that these secondary structures were enriched in the Homininae-specific region of NCYM. Deuteration of NCYM altered the secondary structure at D90 from an α-helix to another structure other than α-helix and ß-strand although this change was within the experimental error range. All four nonsynonymous single-nucleotide polymorphisms (SNPs) in human populations were in this region, and the amino acid alteration in SNP N52S enhanced Myc-nick production. The D90N mutation in NCYM promoted NCYM-mediated MYCN stabilization. Our results reveal the secondary structure of NCYM and demonstrated that the Homininae-specific domain of NCYM is responsible for MYCN stabilization.

Neutron crystallography for the elucidation of enzyme catalysis.

Hydrogen atoms and hydration water molecules in proteins are indispensable for many biochemical processes, especially enzymatic catalysis. The locations of hydrogen atoms in proteins are usually predicted based on X-ray structures, but it is still very difficult to know the ionization states of the catalytic residues, the hydration structure of the protein, and the characteristics of hydrogen-bonding interactions. Neutron crystallography allows the direct observation of hydrogen atoms that play crucial roles in molecular recognition and the catalytic reactions of enzymes. In this review, we present the current status of neutron crystallography in structural biology and recent neutron structural analyses of three enzymes: ascorbate peroxidase, the main protease of severe acute respiratory syndrome coronavirus 2, and copper-containing nitrite reductase.

Reaction mechanism of tetrathionate hydrolysis based on the crystal structure of tetrathionate hydrolase from Acidithiobacillus ferrooxidans.

Tetrathionate hydrolase (4THase) plays an important role in dissimilatory sulfur oxidation in the acidophilic iron- and sulfur-oxidizing bacterium Acidithiobacillus ferrooxidans. The structure of recombinant 4THase from A. ferrooxidans (Af-Tth) was determined by X-ray crystallography to a resolution of 1.95 Å. Af-Tth is a homodimer, and its monomer structure exhibits an eight-bladed ß-propeller motif. Two insertion loops participate in dimerization, and one loop forms a cavity with the ß-propeller region. We observed unexplained electron densities in this cavity of the substrate-soaked structure. The anomalous difference map generated using diffraction data collected at a wavelength of 1.9 Å indicated the presence of polymerized sulfur atoms. Asp325, a highly conserved residue among 4THases, was located near the polymerized sulfur atoms. 4THase activity was completely abolished in the site-specific Af-Tth D325N variant, suggesting that Asp325 plays a crucial role in the first step of tetrathionate hydrolysis. Considering that the Af-Tth reaction occurs only under acidic pH, Asp325 acts as an acid for the tetrathionate hydrolysis reaction. The polymerized sulfur atoms in the active site cavity may represent the intermediate product in the subsequent step.

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.

Structural basis for designing an array of engrailed homeodomains.

Small DNA-binding proteins that target desired sequences have the potential to act as a scaffold for molecular tools such as genome editing. In this study, an engrailed homeodomain (EHD) was chosen and it was evaluated whether it could be used as a molecular module that can connect to itself to recognize a longer target sequence. It was previously shown that two EHDs connected by a linker (EHD2) recognize a target sequence twice as long as that recognized by a single EHD in cells only when Arg53 in each EHD in the tandem protein is mutated to alanine {(EHD[R53A])2}. To investigate the recognition mechanism of (EHD[R53A])2, the crystal structure of the (EHD[R53A])2-DNA complex was determined at 1.6 Šresolution. The individual EHDs were found to adopt the typical homeodomain fold. Most importantly, the base-specific interactions in the major groove necessary for the affinity/specificity of wild-type EHD were preserved in (EHD[R53A])2. Bacterial assays confirmed that the base-specific interactions are retained under cellular conditions. These observations indicate that the R53A mutation only causes a loss of the arginine-phosphate interaction at the protein-DNA interface, which reduces the DNA-binding affinity compared with the wild type. It is therefore concluded that (EHD[R53A])2 precisely recognizes tandem target sites within cells, enabling the individual EHDs to concurrently bind to the target sites with modest binding affinity. This suggests that modulation of the binding activity of each EHD is vital to construct a protein array that can precisely recognize a sequence with multiple target sites.

X-ray crystallographic structural studies of α-amylase I from Eisenia fetida.

The earthworm Eisenia fetida possesses several cold-active enzymes, including α-amylase, ß-glucanase and ß-mannanase. E. fetida possesses two isoforms of α-amylase (Ef-Amy I and II) to digest raw starch. Ef-Amy I retains its catalytic activity at temperatures below 10°C. To identify the molecular properties of Ef-Amy I, X-ray crystal structures were determined of the wild type and of the inactive E249Q mutant. Ef-Amy I has structural similarities to mammalian α-amylases, including the porcine pancreatic and human pancreatic α-amylases. Structural comparisons of the overall structures as well as of the Ca2+-binding sites of Ef-Amy I and the mammalian α-amylases indicate that Ef-Amy I has increased structural flexibility and more solvent-exposed acidic residues. These structural features of Ef-Amy I may contribute to its observed catalytic activity at low temperatures, as many cold-adapted enzymes have similar structural properties. The structure of the substrate complex of the inactive mutant of Ef-Amy I shows that a maltohexaose molecule is bound in the active site and a maltotetraose molecule is bound in the cleft between the N- and C-terminal domains. The recognition of substrate molecules by Ef-Amy I exhibits some differences from that observed in structures of human pancreatic α-amylase. This result provides insights into the structural modulation of the recognition of substrates and inhibitors.

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.

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.

Structural Basis of Mitochondrial Scaffolds by Prohibitin Complexes: Insight into a Role of the Coiled-Coil Region.

The coiled-coil motif mediates subunit oligomerization and scaffolding and underlies several fundamental biologic processes. Prohibitins (PHBs), mitochondrial inner membrane proteins involved in mitochondrial homeostasis and signal transduction, are predicted to have a coiled-coil motif, but their structural features are poorly understood. Here we solved the crystal structure of the heptad repeat (HR) region of PHB2 at 1.7-Å resolution, showing that it assembles into a dimeric, antiparallel coiled-coil with a unique negatively charged area essential for the PHB interactome in mitochondria. Disruption of the HR coiled-coil abolishes well-ordered PHB complexes and the mitochondrial tubular networks accompanying PHB-dependent signaling. Using a proximity-dependent biotin identification (BioID) technique in live cells, we mapped a number of mitochondrial intermembrane space proteins whose association with PHB2 relies on the HR coiled-coil region. Elucidation of the PHB complex structure in mitochondria provides insight into essential PHB interactomes required for mitochondrial dynamics as well as signal transduction.