Flexible Domain Research Articles

Protein disorder in the regulatory control of mitophagy.

Mitophagy is a central component of the mitochondrial quality control machinery, which is necessary for cellular viability and bioenergetics. The E3 ubiquitin ligase CLEC16A (C-type lectin domain containing 16A) forms a tripartite mitophagy regulatory complex together with the E3 ligase RNF41 (ring finger protein 41) and the ubiquitin-specific peptidase USP8 (ubiquitin specific peptidase 8), yet CLEC16A structural/functional domains relevant for mitophagy are unknown. We identify that CLEC16A contains an internal intrinsically disordered region (IDR), which is important for CLEC16A function and stability. IDRs are flexible domains lacking fixed secondary structure and regulate an emerging number of diverse processes, yet they have been largely unstudied in mitophagy. We observe that the internal CLEC16A IDR is essential for CLEC16A degradation and is bound by RNF41 to promote CLEC16A turnover. This IDR also promotes assembly of the CLEC16A-RNF41-USP8 mitophagy regulatory complex. Thus, our study revealed the importance of IDRs in mitophagy via the regulation of CLEC16A abundance by RNF41, opening new structural insights into mitochondrial quality control.

Structural analysis of human ATE1 isoforms and their interactions with Arg-tRNAArg

Posttranslational protein arginylation has been shown as a key regulator of cellular processes in eukaryotes by affecting protein stability, function, and interaction with macromolecules. Thus, the enzyme Arginyltransferase and its targets, are of immense interest to modulate cellular processes in the normal and diseased state. While the study on the effect of this posttranslational modification in mammalian systems gained momentum in the recent times, the detail structures of human ATE1 (hATE1) enzymes has not been investigated so far. Thus, the purpose of this study was to predict the overall structure and the structure function relationship of hATE1 enzyme and its four isoforms. The structure of four ATE1 isoforms were modelled and were docked with 3’end of the Arg-tRNAArg which acts as arginine donor in the arginylation reaction, followed by MD simulation. All the isoforms showed two distinct domains. A compact domain and a somewhat flexible domain as observed in the RMSF plot. A distinct similarity in the overall structure and interacting residues were observed between hATE1-1 and X4 compared to hATE1-2 and 5. While the putative active sites of all the hATE1 isoforms were located at the same pocket, differences were observed in the active site residues across hATE1 isoforms suggesting different substrate specificity. Mining of nsSNPs showed several nsSNPs including cancer associated SNPs with deleterious consequences on hATE1 structure and function. Thus, the current study for the first time shows the structural differences in the mammalian ATE1 isoforms and their possible implications in the function of these proteins. Communicated by Ramaswamy H. Sarma

Development and analysis of a universal label-free micro/nano component for three-channel detection of silver ions, mercury ions, and tetracycline

In this paper, an enzyme-free and label-free fluorescent nanomodule is proposed for rapid, simple and sensitive detection of Ag+, Hg2+ and tetracycline (TC). The strategy is cleverly designed to enable multiple-purpose detection with as little as 31 nt of ssDNA. Both the embedded dye SYBR Green I and the nanomaterial graphene oxide (GO) are able to distinguish single-stranded DNA from double-stranded DNA; thus, the combination of the two instead of using traditional molecular beacon (MB)-labeled fluorophores and quencher groups can effectively reduce the cost of experiments while efficiently reducing the background noise. Performance testing experiments confirmed the stability and selectivity of the platform; the limits of detection (LODs) of Ag+ and Hg2+ were 1.41 nM and 1.79 nM, respectively, and the detection range were within the WHO standards. In addition, only some base sequences in the flexible functional domain of the nanoloop needed to be programmed to build a universal platform, which was feasible using TC as a target. Therefore, the designed nanomodule has the potential to detect various types of targets, such as antibiotics, proteins, and target genes, and has broad application prospects in environmental monitoring, food testing, and disease diagnosis.

Double-headed binding of myosin II to F-actin shows the effect of strain on head structure

Force production in muscle is achieved through the interaction of myosin and actin. Strong binding states in active muscle are associated with Mg·ADP bound to the active site; release of Mg·ADP allows rebinding of ATP and dissociation from actin. Thus, Mg·ADP binding is positioned for adaptation as a force sensor. Mechanical loads on the lever arm can affect the ability of myosin to release Mg·ADP but exactly how this is done is poorly defined. Here we use F-actin decorated with double-headed smooth muscle myosin fragments in the presence of Mg·ADP to visualize the effect of internally supplied tension on the paired lever arms using cryoEM. The interaction of the paired heads with two adjacent actin subunits is predicted to place one lever arm under positive and the other under negative strain. The converter domain is believed to be the most flexible domain within myosin head. Our results, instead, point to the segment of heavy chain between the essential and regulatory light chains as the location of the largest structural change. Moreover, our results suggest no large changes in the myosin coiled coil tail as the locus of strain relief when both heads bind F-actin. The method would be adaptable to double-headed members of the myosin family. We anticipate that the study of actin-myosin interaction using double-headed fragments enables visualization of domains that are typically noisy in decoration with single-headed fragments.

In skeletal muscle and neural crest cells, SMCHD1 regulates biological pathways relevant for Bosma syndrome and facioscapulohumeral dystrophy phenotype.

Many genetic syndromes are linked to mutations in genes encoding factors that guide chromatin organization. Among them, several distinct rare genetic diseases are linked to mutations in SMCHD1 that encodes the structural maintenance of chromosomes flexible hinge domain containing 1 chromatin-associated factor. In humans, its function as well as the impact of its mutations remains poorly defined. To fill this gap, we determined the episignature associated with heterozygous SMCHD1 variants in primary cells and cell lineages derived from induced pluripotent stem cells for Bosma arhinia and microphthalmia syndrome (BAMS) and type 2 facioscapulohumeral dystrophy (FSHD2). In human tissues, SMCHD1 regulates the distribution of methylated CpGs, H3K27 trimethylation and CTCF at repressed chromatin but also at euchromatin. Based on the exploration of tissues affected either in FSHD or in BAMS, i.e. skeletal muscle fibers and neural crest stem cells, respectively, our results emphasize multiple functions for SMCHD1, in chromatin compaction, chromatin insulation and gene regulation with variable targets or phenotypical outcomes. We concluded that in rare genetic diseases, SMCHD1 variants impact gene expression in two ways: (i) by changing the chromatin context at a number of euchromatin loci or (ii) by directly regulating some loci encoding master transcription factors required for cell fate determination and tissue differentiation.

Discovery of SDS-347 as a specific peptide competitive inhibitor of G9a with promising anti-cancer potential

BackgroundG9a is a histone H3K9 methyltransferase enzyme found highly upregulated in many cancers. H3 binds to the rigid I-SET domain and the cofactor, S-adenosyl methionine, binds to the flexible post-SET domain of G9a. Inhibition of G9a is known to inhibit the growth of cancer cell-lines. MethodsRecombinant G9a and H3 were used to develop radioisotope-based inhibitor screening assay. The identified inhibitor was evaluated for isoform selectivity. The mode of enzymatic inhibition was studied by enzymatic assays and bioinformatics. Anti-proliferative activity of the inhibitor was studied in cancer cell lines by utilizing MTT assay. The mechanism of cell death was studied by western blotting and microscopy. ResultsWe developed a robust G9a inhibitor screening assay that led to the discovery of SDS-347 as a potent G9a inhibitor with IC50 of 3.06 μM. It was shown to reduce the levels of H3K9me2 in cell-based assay. The inhibitor was found to be peptide competitive and highly specific as it did not show any significant inhibition of other histone methyltransferases and DNA methyltransferase. Docking studies showed that SDS-347 could form direct bonding interaction with Asp1088 of the peptide-binding site. SDS-347 showed anti-proliferative effect against various cancer cell lines especially the K562 cells. Our data suggested that SDS-347 mediated antiproliferative action via ROS generation, induction of autophagy and apoptosis. ConclusionOverall, the findings of the current study include development of a new G9a inhibitor screening assay and identification of SDS-347, as a novel, peptide competitive and highly specific G9a inhibitor with promising anticancer potential.

Molecular structure of soluble vimentin tetramers

Intermediate filaments (IFs) are essential constituents of the metazoan cytoskeleton. A vast family of cytoplasmic IF proteins are capable of self-assembly from soluble tetrameric species into typical 10–12 nm wide filaments. The primary structure of these proteins includes the signature central ‘rod’ domain of ~ 300 residues which forms a dimeric α-helical coiled coil composed of three segments (coil1A, coil1B and coil2) interconnected by non-helical, flexible linkers (L1 and L12). The rod is flanked by flexible terminal head and tail domains. At present, the molecular architecture of mature IFs is only poorly known, limiting our capacity to rationalize the effect of numerous disease-related mutations found in IF proteins. Here we addressed the molecular structure of soluble vimentin tetramers which are formed by two antiparallel, staggered dimers with coil1B domains aligned (A11 tetramers). By examining a series of progressive truncations, we show that the presence of the coil1A domain is essential for the tetramer formation. In addition, we employed a novel chemical cross-linking pipeline including isotope labelling to identify intra- and interdimeric cross-links within the tetramer. We conclude that the tetramer is synergistically stabilized by the interactions of the aligned coil1B domains, the interactions between coil1A and the N-terminal portion of coil2, and the electrostatic attraction between the oppositely charged head and rod domains. Our cross-linking data indicate that, starting with a straight A11 tetramer, flexibility of linkers L1 and L12 enables ‘backfolding’ of both the coil1A and coil2 domains onto the tetrameric core formed by the coil1B domains. Through additional small-angle X-ray scattering experiments we show that the elongated A11 tetramers dominate in low ionic strength solutions, while there is also a significant structural flexibility especially in the terminal domains.

Histone modifications regulate pioneer transcription factor cooperativity

Pioneer transcription factors have the ability to access DNA in compacted chromatin1. Multiple transcription factors can bind together to a regulatory element in a cooperative way, and cooperation between the pioneer transcription factors OCT4 (also known as POU5F1) and SOX2 is important for pluripotency and reprogramming2–4. However, the molecular mechanisms by which pioneer transcription factors function and cooperate on chromatin remain unclear. Here we present cryo-electron microscopy structures of human OCT4 bound to a nucleosome containing human LIN28B or nMATN1 DNA sequences, both of which bear multiple binding sites for OCT4. Our structural and biochemistry data reveal that binding of OCT4 induces changes to the nucleosome structure, repositions the nucleosomal DNA and facilitates cooperative binding of additional OCT4 and of SOX2 to their internal binding sites. The flexible activation domain of OCT4 contacts the N-terminal tail of histone H4, altering its conformation and thus promoting chromatin decompaction. Moreover, the DNA-binding domain of OCT4 engages with the N-terminal tail of histone H3, and post-translational modifications at H3K27 modulate DNA positioning and affect transcription factor cooperativity. Thus, our findings suggest that the epigenetic landscape could regulate OCT4 activity to ensure proper cell programming.

Yeast PIC-mediator structure with RNA polymerase II C-terminal domain

For transcription initiation, RNA polymerase II (Pol II) forms a preinitiation complex (PIC) that associates with the general coactivator Mediator. Whereas atomic models of the human PIC-Mediator structure have been reported, structures for its yeast counterpart remain incomplete. Here, we present an atomic model for the yeast PIC with core Mediator, including the Mediator middle module that was previously poorly resolved and including subunit Med1 that was previously lacking. We observe three peptide regions containing eleven of the 26 heptapeptide repeats of the flexible C-terminal repeat domain (CTD) of Pol II. Two of these CTD regions bind between the Mediator head and middle modules and form defined CTD-Mediator interactions. CTD peptide 1 binds between the Med6 shoulder and Med31 knob domains, whereas CTD peptide 2 forms additional contacts with Med4. The third CTD region (peptide 3) binds in the Mediator cradle and associates with the Mediator hook. Comparisons with the human PIC-Mediator structure show that the central region in peptide 1 is similar and forms conserved contacts with Mediator, whereas peptides 2 and 3 exhibit distinct structures and Mediator interactions.

An Operational Processing Framework for Spaceborne SAR Formations

The paper proposes a flexible and efficient wavenumber domain processing scheme suited for close formations of low earth orbiting (LEO) synthetic aperture radar (SAR) sensors hosted on micro-satellites or CubeSats. Such systems aim to generate a high-resolution image by combining data acquired by each sensor with a low pulse repetition frequency (PRF). This is usually performed by first merging the different channels in the wavenumber domain, followed by bulk focusing. In this paper, we reverse this paradigm by first upsampling and focusing each acquisition and then combining the focused images to form a high-resolution, unambiguous image. Such a procedure is suited to estimate and mitigate artifacts generated by incorrect positioning of the sensors. An efficient wave–number method is proposed to focus data by adequately coping with the orbit curvature. Two implementations are provided with different quality/efficiency. The image quality in phase preservation, resolution, sidelobes, and ambiguities suppression is evaluated by simulating both point and distributed scatterers. Finally, a demonstration of the capability to compensate for ambiguities due to a small across-track baseline between sensors is provided by simulating a realistic X-band multi-sensor acquisition starting from a stack of COSMO-SkyMed images.

Principles of DNA cleavage in CRISPR-Cas9.

At the core of the CRISPR-Cas9 genome-editing technology, the endonuclease Cas9 introduces site-specific breaks in double-stranded DNA. In this system, the catalysis of target DNA cleavage by the highly flexible HNH domain is undefined, with two possible conformations whose catalytic relevance has remained unmet. Here, extensive molecular simulations - harnessing multi-microsecond molecular dynamics, quantum mechanics and free energy approaches - are combined with solution NMR and DNA cleavage assays to establish how the HNH nuclease is activated and cleaves the target DNA. We show that the conformation of the active state is critically dependent on the presence of Mg2+, unveiling its cardinal structural role in the organization of the active site. Solution NMR, DNA cleavage assays and molecular simulations of the Mg2+-bound HNH report on the formation of this active state, and consistently show that the protonation state of catalytic H840 is strongly affected by residue mutations within the active site. Finally, quantum mechanical simulations establish that DNA cleavage occurs through the identified active state, showing that the catalysis is activated by H840 and completed by K866, in line with DNA cleavage experiments. Overall, high-level computations, supported by experimental evaluation, resolve the catalytic mechanism and which of the known HNH conformations is responsible for target DNA cleavage in CRISPR-Cas9. This information helps enhancing the catalytic efficiency of Cas9 and its specificity, furthering the development of genome-editing tools.

Understanding the effects of higher-order assembly on the structure of the sulfite reductase.

The redox state of sulfur determines its bioavailbility and its reduction to sulfide is required for assimilation of sulfur into amino acids and cofactors.The NADPH-dependent assimilatory sulfite reductase (SiR) is a metabolic enzyme that performs the six-electron reduction of sulfite to sulfide. Escherichia coli SiR is composed of eight flavoprotein (SiRFP, ⍺) and four hemoprotein (SiRHP, β) subunits that associate in α8:β4 holoenzyme. This dodecameric assembly is unique because SiRs from other sulfur reducing organisms are dimers of reductase and oxidase subunits. Both SiR subunits are modular. SiRFP is a fusion between flavodoxin domain and a ferredoxin-NADP+ reductase domain that assembles through its 52 N-terminal amino acids into an octamer. SiRHP is a monomeric metalloenzyme that houses the active site. Despite almost 30 years of effort across many laboratories, structures of SiR complexes have remained recalcitrant to X-ray crystallography and cryo-EM because of its structural heterogeneity aising from intrinsically disordered regions throughout the complex, including the flexible linker joining SiRFP's flavin binding domain. Thus we do not know how the domains assemble, which leaves a gap in understanding about how these domains coordinate to perform electron transfer. Here, we use neutron contrast variation (NCV) and small angle neutron scattering (SANS) to observe the relative subunit positioning within the SiR complex. To better understand the electron transfer mechanism, we have generated conformationally restricted variant of SiRFP that either locks its flavin binding domains in an open conformation by shortening the linker between domains, or in closed conformation by engineering disulfide bonds. SANS and NCV study reveal SiR's asymmetric complex, and the resulting maps supports a redundant, cis/trans mechanism of electron transfer between the reductase subunits as well as between the tightly or transiently bound reductase and oxidase domains.

Defining the structure and orientation of the N-terminal domain of cardiac troponin T in relationship to actin.

Cardiac troponin T (cTnT) serves as the tropomyosin (Tm) binding subunit of the troponin complex and, when mutated, it often leads to pathologic cardiac remodeling observed in patients with hypertrophic and dilated cardiomyopathies. Part of the N-terminus of cTnT is responsible for positioning Tm along the actin groove and it immediately flanks the Tm overlap domain. Understanding the molecular mechanisms underlying these observations has been hindered by the absence of a high-resolution structure in the highly flexible N-terminal domain. A recently published cryo-electron-microscopy structure shows interactions between the N-terminus of cTnT, around amino acid 87, and the F-actin backbone; however, this interaction was not observed in calcium saturated conditions. Here, we employ single-donor dual-acceptor time-resolved fluorescence resonance energy transfer (TR-FRET) to probe the positioning of the cTnT N-terminal extension in fully reconstituted cardiac thin filaments (CTF). We hypothesize the N-terminal tail interacts with F-actin in the −Ca2+ condition aiding in stabilizing Tm in the blocked position. Conversely, we hypothesize that the that the N-terminal tail is “fly-casting” and not interacting with F-actin in +Ca2+ condition. To test this, a donor probe (IAEDANS) was placed on either the Cys-substituted residues N100C, N73C and M60C of cTnT and an acceptor probe (DABMI) was placed on the endogenous Cys-374 of actin. Preliminary data revealed two measurable discrete distances for all residues, one short and one long, indicating that FRET occurred with two actin monomers. Initial results suggest that the N-terminal tail is interacting with F-actin in high and low calcium conditions. Continuing work aims to probe additional sites on the N-terminus, specifically A20C and A2C. Resolving the structure and orientation of the N-terminal domain of cTnT is critical to understanding the regulatory mechanisms of the CTF.

Distinct conformational changes occur within the intrinsically unstructured pro-domain of pro-Nerve Growth Factor in the presence of ATP and Mg2.

Nerve growthfactor (NGF), the prototypical neurotrophic factor, is involved in the maintenance and growth of specific neuronal populations, whereas its precursor, proNGF, is involved in neuronal apoptosis. Binding of NGF or proNGF to TrkA, p75NTR , and VP10p receptors triggers complex intracellular signaling pathways that can be modulated by endogenous small-molecule ligands. Here, we show by isothermal titration calorimetry and NMR that ATP binds to the intrinsically disordered pro-peptide of proNGF with a micromolar dissociation constant. We demonstrate that Mg2+ , known to play a physiological role in neurons, modulates the ATP/proNGF interaction. An integrative structural biophysics analysis by small angle X-ray scattering and hydrogen-deuterium exchange mass spectrometry unveils that ATP binding induces a conformational rearrangement of the flexible pro-peptide domain of proNGF. This suggests that ATP may act as an allosteric modulator of the overall proNGF conformation, whose likely distinct biological activity may ultimately affect its physiological homeostasis.

Interdomain dynamics in human Replication Protein A regulates kinetics and thermodynamics of its binding to ssDNA.

Human Replication Protein A (hRPA) is a multidomain protein that interacts with ssDNA intermediates to provide the latter much-needed stability during DNA metabolism and maintain genomic integrity. Although the ssDNA organization with hRPA was studied recently through experimental means, characterizing the underlying mechanism at the atomic level remains challenging because of the dynamic domain architecture of hRPA and poorly understood heterogeneity of ssDNA-protein interactions. Here, we used a computational framework, precisely tailored to capture protein-ssDNA interactions, and investigated the binding of hRPA with a 60 nt ssDNA. Two distinct binding mechanisms are realized based on the hRPA domain flexibility. For a rigid domain architecture of hRPA, ssDNA binds sequentially with hRPA domains, resulting in slow association kinetics. The binding pathway involves the formation of stable and distinct intermediate states. On contrary, for a flexible domain architecture of hRPA, ssDNA binds synergistically to the A and B domains followed by the rest of hRPA. The domain dynamics in hRPA alleviates the free energy cost of domain orientation necessary for specific binding with ssDNA, leading to fast association kinetics along a downhill binding free energy landscape. An ensemble of free energetically degenerate intermediate states is encountered that makes it arduous to characterize them structurally. An excellent match between our results with the available experimental observations provides new insights into the rich dynamics of hRPA binding to ssDNA and in general paves the way to investigate intricate details of ssDNA-protein interactions, crucial for cellular functioning.

Forecasting the July Precipitation over the Middle-Lower Reaches of the Yangtze River with a Flexible Statistical Model

The multiple regression method is still an important tool for establishing precipitation forecast models with a lead time of one season. This study developed a flexible statistical forecast model for July precipitation over the middle-lower reaches of the Yangtze River (MLYR) based on the prophase winter sea surface temperature (SST). According to the characteristics of observed samples and related theoretical knowledge, some special treatments (i.e., more flexible and better-targeted methods) were introduced in the forecast model. These special treatments include a flexible MLYR domain definition, the extraction of indicative signals from the SST field, artificial samples, and the amplification of abnormal precipitation. Rolling forecast experiments show that the linear correlation between prediction and observation is around 0.5, more than half of the abnormal precipitation years can be successfully predicted, and there is no contradictory prediction of the abnormal years. These results indicate that the flexible statistical forecast model is valuable in real-life applications. Furthermore, sensitivity experiments show that forecast skills without these special treatments are obviously decreased. This suggests that forecast models can benefit from using statistical methods in a more flexible and better-targeted way.

Abstract 2675: The Dnase1L3 flexible C-terminal domain and altered charges in the actin recognition site result in broad substrate diversity and resistance to actin inhibition

Abstract 2675: The Dnase1L3 flexible C-terminal domain and altered charges in the actin recognition site result in broad substrate diversity and resistance to actin inhibition

Two-Layer Generation Expansion Planning Based on Flexibility Balance

Reasonable planning of flexible power sources is crucial for the reliable operation of new power systems based on new energy sources. However, in the long-term planning, how to consider the short-term ramping capability of the flexible power supply to meet the capacity demand of the net load and the flexible ramping requirement at the same time is still an unsolved problem. Therefore, this paper conducts research on generation expansion planning based on flexibility balance. Firstly, the flexibility balance are modeled based on the flexible supply domain and the net load fluctuation domain, which are used as the flexible supply and demand constraints in the planning stage. Then, a two-layer expansion planning model is constructed, including two modules of investment decision and production simulation. Flexibility index is proposed to quantify the system’s ability to adjust at different levels of payload, and incorporated into the planning model for iterative optimization. Finally, an example is verified on the improved IEEE-39 node system. The test results show that the long-term generation expansion planning scheme based on the balance of flexible supply and demand can effectively meet the net load demand and its ramping demand.

A structural perspective of liver X receptors.

Liver X receptors α and β are members of the nuclear receptor family, which comprise a flexible N-terminal domain, a DNA binding domain, a hinge linker, and a ligand binding domain. Liver X receptors are important regulators of cholesterol and lipid homeostasis by controlling the transcription of numerous genes. Key to their transcriptional role is synergetic interaction among the domains. DNA binding domain binds on DNA; ligand binding domain is a crucial switch to control the transcription activity through conformational change caused by ligand binding. The Liver X receptors form heterodimers with retinoid X receptor and then the liganded heterodimer may recruit other necessary transcription components to form an active transcription complex.

Aircraft Trajectory Prediction Based on Bayesian Optimised Temporal Convolutional Network–Bidirectional Gated Recurrent Unit Hybrid Neural Network

Efficient and accurate flight trajectory prediction is a key technology for promoting intelligent and informative air traffic management and improving the operational capabilities and predictability of air traffic. To address the problems in extracting hidden information from historical trajectory information, the approach must accurately select high-dimensional features related to the prediction target and overcome the short-term memory of the time series. Herein, we present a novel trajectory prediction model based on a dual-self-attentive (DSA)-temporal convolutional network (TCN)-bidirectional gated recurrent unit (BiGRU) neural network. In this model, the TCN provides highly stable training, high parallelism, and a flexible perceptual domain. The self-attentive mechanism of the TCN structure can focus on features that contribute the most to the output. After the TCN, the BiGRU network combined with the self-attentive mechanism is used to further bidirectionally mine the connections between the features and outputs of the trajectory sequence, and a Bayesian algorithm is used to optimise the hyperparameters of the model for optimal performance. A comparison and validation based on current well-known neural network models (i.e., CNN, TCN, GRU, and their variants) shows that the DSA-TCN-BiGRU model based on Bayesian hyperparameter optimisation has the best performance. Therefore, the improved predictive model is applicable and valuable, providing a basis for future decision trajectory-based operations.