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.

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.

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).

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.

Nanoscopic changes in the lattice structure of striated muscle sarcomeres involved in the mechanism of spontaneous oscillatory contraction (SPOC).

Muscles perform a wide range of motile functions in animals. Among various types are skeletal and cardiac muscles, which exhibit a steady auto-oscillation of force and length when they are activated at an intermediate level of contraction. This phenomenon, termed spontaneous oscillatory contraction or SPOC, occurs devoid of cell membranes and at fixed concentrations of chemical substances, and is thus the property of the contractile system per se. We have previously developed a theoretical model of SPOC and proposed that the oscillation emerges from a dynamic force balance along both the longitudinal and lateral axes of sarcomeres, the contractile units of the striated muscle. Here, we experimentally tested this hypothesis by developing an imaging-based analysis that facilitates detection of the structural changes of single sarcomeres at unprecedented spatial resolution. We found that the sarcomere width oscillates anti-phase with the sarcomere length in SPOC. We also found that the oscillatory dynamics can be altered by osmotic compression of the myofilament lattice structure of sarcomeres, but they are unchanged by a proteolytic digestion of titin/connectin-the spring-like protein that provides passive elasticity to sarcomeres. Our data thus reveal the three-dimensional mechanical dynamics of oscillating sarcomeres and suggest a structural requirement of steady auto-oscillation.

Dynamic Properties of Human α-Synuclein Related to Propensity to Amyloid Fibril Formation.

α-Synuclein (αSyn) is an intrinsically disordered protein that can form amyloid fibrils. Fibrils of αSyn are implicated with the pathogenesis of Parkinson's disease and other synucleinopathies. Elucidating the mechanism of fibril formation of αSyn is therefore important for understanding the mechanism of the pathogenesis of these diseases. Fibril formation of αSyn is sensitive to solution conditions, suggesting that fibril formation of αSyn arises from the changes in its inherent physico-chemical properties, particularly its dynamic properties because intrinsically disordered proteins such as αSyn utilize their inherent flexibility to function. Characterizing these properties under various conditions should provide insights into the mechanism of fibril formation. Here, using the quasielastic neutron scattering and small-angle x-ray scattering techniques, we investigated the dynamic and structural properties of αSyn under the conditions, where mature fibrils are formed (pH 7.4 with a high salt concentration), where clumping of short fibrils occurs (pH 4.0), and where fibril formation is not completed (pH 7.4). The small-angle x-ray scattering measurements showed that the extended structures at pH 7.4 with a high salt concentration become compact at pH 4.0 and 7.4. The quasielastic neutron scattering measurements showed that both intra-molecular segmental motions and local motions such as side-chain motions are enhanced at pH 7.4 with a high salt concentration, compared to those at pH 7.4 without salt, whereas only the local motions are enhanced at pH 4.0. These results imply that fibril formation of αSyn requires not only the enhanced local motions but also the segmental motions such that proper inter-molecular interactions are possible.

Effects of the cardiomyopathy-causing E244D mutation of troponin T on the structures of cardiac thin filaments studied by small-angle X-ray scattering.

Small-angle X-ray scattering experiments were carried out to investigate the structural changes of cardiac thin filaments induced by the cardiomyopathy-causing E244D mutation in troponin T (TnT). We examined native thin filaments (NTF) from a bovine heart, reconstituted thin filaments containing human cardiac wild-type Tn (WTF), and filaments containing the E244D mutant of Tn (DTF), in the absence and presence of Ca2+. Analysis by model calculation showed that upon Ca2+-activation, tropomyosin (Tm) and Tn in the WTF and NTF moved together in a direction to expose myosin-binding sites on actin. On the other hand, Tm and Tn of the DTF moved in the opposite directions to each other upon Ca2+-activation. These movements caused Tm to expose more myosin-binding sites on actin than the WTF, suggesting that the affinity of myosin for actin is higher for the DTF. Thus, the mutation-induced structural changes in thin filaments would increase the number of myosin molecules bound to actin compared with the WTF, resulting in the force enhancement observed for the E244D mutation.

Modulation of the picosecond dynamics of troponin by the cardiomyopathy-causing mutation K247R of troponin T observed by quasielastic neutron scattering.

Troponin (Tn), consisting of three subunits (TnC, TnI, and TnT), regulates cardiac muscle contraction in a Ca2+-dependent manner. Various point mutations of human cardiac Tn are known to cause familial hypertrophic cardiomyopathy due to aberration of the regulatory function. In this study, we investigated the effects of one of these mutations, K247R of TnT, on the picosecond dynamics of the Tn core domain (Tn-CD), consisting of TnC, TnI and TnT2 (183-288 residues of TnT), by carrying out the quasielastic neutron scattering measurements on the reconstituted Tn-CD containing either the wild-type TnT2 (wtTn-CD) or the mutant TnT2 (K247R-Tn-CD) in the absence and presence of Ca2+. It was found that Ca2+-binding to the wtTn-CD decreases the residence time of atomic motions in the Tn-CD with slight changes in amplitudes, suggesting that the regulatory function mainly requires modulation of frequency of atomic motions. On the other hand, the K247R-Tn-CD shows different dynamic behavior from that of the wtTn-CD both in the absence and presence of Ca2+. In particular, the K247R-Tn-CD exhibits a larger amplitude than the wtTn-CD in the presence of Ca2+, suggesting that the mutant can explore larger conformational space than the wild-type. This increased flexibility should be relevant to the functional aberration of this mutant.

Ligation-Dependent Picosecond Dynamics in Human Hemoglobin As Revealed by Quasielastic Neutron Scattering.

Hemoglobin, the vital O2 carrier in red blood cells, has long served as a classic example of an allosteric protein. Although high-resolution X-ray structural models are currently available for both the deoxy tense (T) and fully liganded relaxed (R) states of hemoglobin, much less is known about their dynamics, especially on the picosecond to subnanosecond time scales. Here, we investigate the picosecond dynamics of the deoxy and CO forms of human hemoglobin using quasielastic neutron scattering under near physiological conditions in order to extract the dynamics changes upon ligation. From the analysis of the global motions, we found that whereas the apparent diffusion coefficients of the deoxy form can be described by assuming translational and rotational diffusion of a rigid body, those of the CO form need to involve an additional contribution of internal large-scale motions. We also found that the local dynamics in the deoxy and CO forms are very similar in amplitude but are slightly lower in frequency in the former than in the latter. Our results reveal the presence of rapid large-scale motions in hemoglobin and further demonstrate that this internal mobility is governed allosterically by the ligation state of the heme group.

Nonlinear force-length relationship in the ADP-induced contraction of skeletal myofibrils.

The regulatory mechanism of sarcomeric activity has not been fully clarified yet because of its complex and cooperative nature, which involves both Ca(2+) and cross-bridge binding to the thin filament. To reveal the mechanism of regulation mediated by the cross-bridges, separately from the effect of Ca(2+), we investigated the force-sarcomere length (SL) relationship in rabbit skeletal myofibrils (a single myofibril or a thin bundle) at SL > 2.2 microm in the absence of Ca(2+) at various levels of activation by exogenous MgADP (4-20 mM) in the presence of 1 mM MgATP. The individual SLs were measured by phase-contrast microscopy to confirm the homogeneity of the striation pattern of sarcomeres during activation. We found that at partial activation with 4-8 mM MgADP, the developed force nonlinearly depended on the length of overlap between the thick and the thin filaments; that is, contrary to the maximal activation, the maximal active force was generated at shorter overlap. Besides, the active force became larger, whereas this nonlinearity tended to weaken, with either an increase in [MgADP] or the lateral osmotic compression of the myofilament lattice induced by the addition of a macromolecular compound, dextran T-500. The model analysis, which takes into account the [MgADP]- and the lattice-spacing-dependent probability of cross-bridge formation, was successfully applied to account for the force-SL relationship observed at partial activation. These results strongly suggest that the cross-bridge works as a cooperative activator, the function of which is highly sensitive to as little as