Structural Basis of Nucleic Acid Recognition and 6mA Demethylation by Caenorhabditis elegans NMAD-1A.

N6-methyladenine (6mA) of DNA is an emerging epigenetic mark in the genomes of Chlamydomonas, Caenorhabditis elegans, and mammals recently. Levels of 6mA undergo drastic fluctuation and thus affect fertility during meiosis and early embryogenesis. Here, we showed three complex structures of 6mA demethylase C. elegans NMAD-1A, a canonical isoform of NMAD-1 (F09F7.7). Biochemical results revealed that NMAD-1A prefers 6mA Bubble or Bulge DNAs. Structural studies of NMAD-1A revealed an unexpected "stretch-out" conformation of its Flip2 region, a conserved element that is usually bent over the catalytic center to facilitate substrate base flipping in other DNA demethylases. Moreover, the wide channel between the Flip1 and Flip2 of the NMAD-1A explained the observed preference of NMAD-1A for unpairing substrates, of which the flipped 6mA was primed for catalysis. Structural analysis and mutagenesis studies confirmed that key elements such as carboxy-terminal domain (CTD) and hypothetical zinc finger domain (ZFD) critically contributed to structural integrity, catalytic activity, and nucleosome binding. Collectively, our biochemical and structural studies suggest that NMAD-1A prefers to regulate 6mA in the unpairing regions and is thus possibly associated with dynamic chromosome regulation and meiosis regulation.

The Rigid Core and Flexible Surface of Amyloid Fibrils Probed by Magic-Angle-Spinning NMR Spectroscopy of Aromatic Residues.

Aromatic side chains are important reporters of the plasticity of proteins, and often form important contacts in protein-protein interactions. We studied aromatic residues in the two structurally homologous cross-ß amyloid fibrils HET-s, and HELLF by employing a specific isotope-labeling approach and magic-angle-spinning NMR. The dynamic behavior of the aromatic residues Phe and Tyr indicates that the hydrophobic amyloid core is rigid, without any sign of "breathing motions" over hundreds of milliseconds at least. Aromatic residues exposed at the fibril surface have a rigid ring axis but undergo ring flips on a variety of time scales from nanoseconds to microseconds. Our approach provides direct insight into hydrophobic-core motions, enabling a better evaluation of the conformational heterogeneity generated from an NMR structural ensemble of such amyloid cross-ß architecture.

The effect of spin-lattice relaxation on DEER background decay.

The common approach to background removal in double electron-electron resonance (DEER) measurements on frozen solutions with a three-dimensional homogeneous distribution of doubly labeled biomolecules is to fit the background to an exponential decay function. Excluded volume effects or distribution in a dimension lower than three, such as proteins in a membrane, can lead to a stretched exponential decay. In this work, we show that in cases of spin labels with short spin-lattice relaxation time, up to an order of magnitude longer than the DEER trace length, relevant for metal-based spin labels, spin flips that take place during the DEER evolution time affect the background decay shape. This was demonstrated using a series of temperature-dependent DEER measurements on frozen solutions of a nitroxide radical, a Gd(III) complex, Cu(II) ions, and a bis-Gd(III) model complex. As expected, the background decay was exponential for the nitroxide, whereas deviations were noted for Gd(III) and Cu(II). Based on the theoretical approach of Keller et al. (Phys. Chem. Chem. Phys. 21 (2019) 8228-8245), which addresses the effect of spin-lattice relaxation-induced spin flips during the evolution time, we show that the background decay can be fitted to an exponent including a linear and quadratic term in t, which is the position of the pump pulse. Analysis of the data in terms of the probability of spontaneous spin flips induced by spin-lattice relaxation showed that this approach worked well for the high temperature range studied for Gd(III) and Cu(II). At the low temperature range, the spin flips that occured during the DEER evolution time for Gd(III) exceeded the measured spin-lattice relaxation rate and include contributions from spin flips due to another mechanisms, most likely nuclear spin diffusion.

Triptolide-mediated downregulation of FLIPS in hepatoma cells occurs at the post-transcriptional level independently of proteasome-mediated pathways.

Cellular c-FLIP prevents apoptosis mediated by death receptor through inhibiting activation of caspase-8. Therefore, when c-FLIP is downregulated or eliminated, caspase-8 activation is promoted, and death receptor ligand-induced apoptosis is activated. It was reported that triptolide (TPL) sensitized tumor cells to TNF-α-induced apoptosis by blocking TNF-α-induced activation of NF-κB and transcription of c-IAP1 and c-IAP2. However, the effect of TPL on basal c-FLIP expression was not understood. In this study, we found that the combination of TNF-α and TPL accelerated apoptosis in human hepatocellular carcinoma cells and TNF-α-induced elevated as well as basal level of FLIPS protein were downregulated by TPL. Additionally, we demonstrated that the basal level of FLIPS in Huh7 cells was continuously downregulated following the incubation of TPL and downregulated more when dosage of TPL for treatment was increased. Subsequently, we showed that TPL reduced FLIPS level in a transcription- and degradation-independent mechanism. Our findings suggest that TPL induces loss of FLIPS at the post-transcriptional level independently of proteasome-mediated pathway, an additional mechanism of TPL sensitizing cancer cells to TNF-α-induced apoptosis.

GPCR large-amplitude dynamics by 19F-NMR of aprepitant bound to the neurokinin 1 receptor.

Comparisons of G protein-coupled receptor (GPCR) complexes with agonists and antagonists based on X-ray crystallography and cryo-electron microscopy structure determinations show differences in the width of the orthosteric ligand binding groove over the range from 0.3 to 2.9 Å. Here, we show that there are transient structure fluctuations with amplitudes up to at least 6 Å. The experiments were performed with the neurokinin 1 receptor (NK1R), a GPCR of class A that is involved in inflammation, pain, and cancer. We used 19F-NMR observation of aprepitant, which is an approved drug that targets NK1R for the treatment of chemotherapy-induced nausea and vomiting. Aprepitant includes a bis-trifluoromethyl-phenyl ring attached with a single bond to the core of the molecule; 19F-NMR revealed 180° flipping motions of this ring about this bond. In the picture emerging from the 19F-NMR data, the GPCR transmembrane helices undergo large-scale floating motions in the lipid bilayer. The functional implication is of extensive promiscuity of initial ligand binding, primarily determined by size and shape of the ligand, with subsequent selection by unique interactions between atom groups of the ligand and the GPCR within the binding groove. This second step ensures the wide range of different efficacies documented for GPCR-targeting drugs. The NK1R data also provide a rationale for the observation that diffracting GPCR crystals are obtained for complexes with only very few of the ligands from libraries of approved drugs and lead compounds that bind to the receptors.

Experimental observation of carousel-like phason flips in the decagonal quasicrystal Al60Cr20Fe10Si10.

Quasicrystals have special crystal structures with long-range order, but without translational symmetry. Unexpectedly, carousel-like successive flippings of groups of atoms inside the ∼2 nm decagonal structural subunits of the decagonal quasicrystal Al60Cr20Fe10Si10 were directly observed using in situ high-temperature high-resolution transmission electron microscopy imaging. The observed directionally successive phason flips occur mainly clockwise and occasionally anticlockwise. The origin of these directional phason flips is analyzed and discussed.

OCRE Domains of Splicing Factors RBM5 and RBM10: Tyrosine Ring-Flip Frequencies Determined by Integrated Use of 1 H NMR Spectroscopy and Molecular Dynamics Simulations.

The 55-residue OCRE domains of the splicing factors RBM5 and RBM10 contain 15 tyrosines in compact, globular folds. At 25 °C, all 15 tyrosines show symmetric 1 H NMR spectra, with averaged signals for the pairs of δ- and ϵ-ring hydrogens. At 4 °C, two tyrosines were identified as showing 1 H NMR line-broadening due to lowered frequency of the ring-flipping. For the other 13 tyrosine rings, it was not evident, from the 1 H NMR data alone, whether they were either all flipping at high frequencies, or whether slowed flipping went undetected due to small chemical-shift differences between pairs of exchanging ring hydrogen atoms. Here, we integrate 1 H NMR spectroscopy and molecular dynamics (MD) simulations to determine the tyrosine ring-flip frequencies. In the RBM10-OCRE domain, we found that, for 11 of the 15 tyrosines, these frequencies are in the range 2.0×106 to 1.3×108  s-1 , and we established an upper limit of <1.0×106  s-1 for the remaining four residues. The experimental data and the MD simulation are mutually supportive, and their combined use extends the analysis of aromatic ring-flip events beyond the limitations of routine 1 H NMR line-shape analysis into the nanosecond frequency range.

Backbone and side-chain chemical shift assignments of a cellular FLICE-inhibitory protein (c-FLIPS).

Cellular FLICE-inhibitory protein (c-FLIP), which is involved in regulating the apoptosis of the extrinsic cell death pathway contains two death effector domains (DED). There are several splicing variants including short-form (c-FLIPS) and long-form (c-FLIPL). The death-inducing signaling complex (DISC) initiates apoptosis and programmed necrosis, DISC assembly and activation are regulated by c-FLIP. Here we report the NMR chemical shift assignments of c-FLIPs, which pave the way for investigating the molecular basis of the anti-apoptotic function of c-FLIPS.

Phason-flips refinement of and multiple-scattering correction for the d-AlCuRh quasicrystal.

The origin of the characteristic bias observed in a logarithmic plot of the calculated and measured intensities of diffraction peaks for quasicrystals has not yet been established. Structure refinement requires the inclusion of weak reflections; however, no structural model can properly describe their intensities. For this reason, detailed information about the atomic structure is not available. In this article, a possible cause for the characteristic bias, namely the lattice phason flip, is investigated. The derivation of the structure factor for a tiling with inherent phason flips is given and is tested for the AlCuRh decagonal quasicrystal. Although an improvement of the model is reported, the bias remains. A simple correction term involving a redistribution of the intensities of the peaks was tested, and successfully removed the bias from the diffraction data. This new correction is purely empirical and only mimics the effect of multiple scattering. A comprehensive study of multiple scattering requires detailed knowledge of the diffraction experiment geometry.

Dynamic nuclear polarization via the cross effect and thermal mixing: B. Energy transport.

The fundamental process of dynamic nuclear polarization (DNP) via the cross effect (CE) and thermal mixing (TM) is a triple spin flip, in which two interacting electron spins and a nuclear spin interacting with one of these electron spins flip together. In the previous article (Wenckebach, 2018) these triple spin flips were treated by first determining the eigenstates of the two interacting electron spins exactly and next investigating transitions involving these exact eigenstates and the nuclear spin states. It was found that two previously developed approaches-the scrambled states approach and the fluctuating field approach-are just two distinct limiting cases of this more general approach. It was also shown that triple spin flips constitute a single process causing two flows of energy: a flow originating in the electron Zeeman energy and a flow originating in the mutual interactions between the electron spins. In order to render their definitions more precise, the former flow was denoted as the CE and the latter as TM. In this article the treatment is extended to a glass containing NI equivalent nuclear spins I=12 and NS randomly distributed and oriented electron spins S=12. Rate equations are derived for the two flows of energy to the nuclear spins. The flow originating in the electron Zeeman energy-i.e. the CE-is found to lead to the same stationary state as was previously predicted by the scrambled states approach, though the rate may be smaller due to limitations imposed by conservation of energy. The flow originating in the mutual interactions between the electron spins-i.e. TM-is found to involve the full spectrum of the mutual interactions between the electron spins, while the fluctuating field approach only accounts for the component of this spectrum at the nuclear magnetic resonance (NMR) frequency. Still, TM is found to induce equal spin temperature for different nuclear spin species during nuclear spin-lattice relaxation and, at least in some cases also during polarization. It is also confirmed that TM couples local nuclear spins near the electron spins so strongly to the mutual interactions between electron spins, that they may constitute a single energy reservoir (Cox et al., 1973). Hence such local nuclear spins may have to be included in treatments of the dynamics of the electron spins.

Optimal Isotope Labeling of Aromatic Amino Acid Side Chains for NMR Studies of Protein Dynamics.

Aromatic side chains in proteins are often directly evolved in stabilizing the hydrophobic core, protein binding, or enzymatic activity. They are also responsible for specific local dynamic processes, such as histidine tautomerization or ring flips. Despite their importance, they are often not targeted directly by NMR spectroscopy, because of spectroscopic complications and challenges. This chapter addresses state-of-the-art site-selective 13C-labeling methods for aromatic side chains, and describes how they solve several of the spectroscopic issues. A special emphasis is put on thereby enabled protein dynamics experiments of aromatic side chains.

Dynamic nuclear polarization via the cross effect and thermal mixing: A. The role of triple spin flips.

In dynamic nuclear polarization (DNP) via the cross effect (CE) and thermal mixing (TM) a microwave field first reduces the polarization of some electron spins, so the electron spin system deviates from thermal equilibrium with the lattice. Next, the mutual interactions combine with their interaction with the nuclear spins to transform this deviation into nuclear spin polarization. Two approaches were introduced to describe the latter process. The fluctuating field approach considers the electron spins to fluctuate between their up and down states due to their mutual interactions. This results in a classical fluctuating field at the position of the nuclear spins, and the component of this field at the NMR frequency induces flips of the nuclear spins. The scrambled states approach considers the electron and nuclear spin states to be mixed by the hyperfine and super-hyperfine interaction. Next the mutual interaction between the electron spins induces transitions between these mixed states and thus flips nuclear spins. Some authors considered the fluctuating field approach and the scrambled states approach to be just two equivalent methods to describe exactly the same process. Other authors considered the two approaches to describe two separate processes, the former exchanging electron interaction energy, the latter transferring differences of electron spin polarization to the nuclear spins. The present work introduces a generalized approach that first calculates the mixing of electron spin states exactly. Next it considers the hyperfine or super-hyperfine interaction to induce transitions involving these mixed states and the nuclear spin states. It is found that the scrambled states approach and the fluctuating field approach are neither fully equivalent, nor completely independent processes, but rather represent two distinct limits of a single process. The former corresponding to very weak mutual interactions between electron spins and the latter to very strong mutual interactions. It extends the treatment to the whole range of mutual interactions and shows that this single process simultaneously exchanges electron Zeeman energy and electron interaction energy with the nuclear spins. Expressions for these two flows as a function of the strength of the mutual interaction are derived.

Comprehensive analysis of sequences of a protein switch.

Switches form a special class of proteins that dramatically change their three-dimensional structures upon a small perturbation. One possible perturbation that we explore is that of a single point mutation. Building on the pioneering experimental work of Alexander et al. (Alexander et al. PNAS, 2007; 104,11963-11968) that determines switch sequences between α and α+ß folds we conduct a comprehensive sequence sampling by a Markov Chain with multiple fitness criteria to identify new switches given the experimental folds. We screen for switch sequences using a combination of contact potential, secondary structure prediction, and finally molecular dynamics simulations. Statistical properties of switch sequences are discussed and illustrated to be most sensitive to mutation at the N- and C- termini of the switch protein. Based on this analysis, a particularly stable putative switch pair is identified and proposed for further experimental analysis.

Integrating the invisible fabric of nature into fisheries management.

Overfishing and environmental change have triggered many severe and unexpected consequences. As existing communities have collapsed, new ones have become established, fundamentally transforming ecosystems to those that are often less productive for fisheries, more prone to cycles of booms and busts, and thus less manageable. We contend that the failure of fisheries science and management to anticipate these transformations results from a lack of appreciation for the nature, strength, complexity, and outcome of species interactions. Ecologists have come to understand that networks of interacting species exhibit nonlinear dynamics and feedback loops that can produce sudden and unexpected shifts. We argue that fisheries science and management must follow this lead by developing a sharper focus on species interactions and how disrupting these interactions can push ecosystems in which fisheries are embedded past their tipping points.

Inhibitor of apoptosis proteins (IAPs) as regulatory factors of hepatic apoptosis.

IAPs are a group of regulatory proteins that are structurally related. Their conserved homologues have been identified in various organisms. In human, eight IAP members have been recognized based on baculoviral IAP repeat (BIR) domains. IAPs are key regulators of apoptosis, cytokinesis and signal transduction. The antiapoptotic property of IAPs depends on their professional role for caspases. IAPs are functionally non-equivalent and regulate effector caspases through distinct mechanisms. IAPs impede apoptotic process via membrane receptor-dependent (extrinsic) cascade and mitochondrial dependent (intrinsic) pathway. IAP-mediated apoptosis affects the progression of liver diseases. Therapeutic options of liver diseases may depend on the understanding toward mechanisms of the IAP-mediated apoptosis.