Structural basis of PETISCO complex assembly during piRNA biogenesis in C. elegans.

Piwi-interacting RNAs (piRNAs) constitute a class of small RNAs that bind PIWI proteins and are essential to repress transposable elements in the animal germline, thereby promoting genome stability and maintaining fertility. C. elegans piRNAs (21U RNAs) are transcribed individually from minigenes as precursors that require 5' and 3' processing. This process depends on the PETISCO complex, consisting of four proteins: IFE-3, TOFU-6, PID-3, and ERH-2. We used biochemical and structural biology approaches to characterize the PETISCO architecture and its interaction with RNA, together with its effector proteins TOST-1 and PID-1. These two proteins define different PETISCO functions: PID-1 governs 21U processing, whereas TOST-1 links PETISCO to an unknown process essential for early embryogenesis. Here, we show that PETISCO forms an octameric assembly with each subunit present in two copies. Determination of structures of the TOFU-6/PID-3 and PID-3/ERH-2 subcomplexes, supported by in vivo studies of subunit interaction mutants, allows us to propose a model for the formation of the TOFU-6/PID-3/ERH-2 core complex and its functionality in germ cells and early embryos. Using NMR spectroscopy, we demonstrate that TOST-1 and PID-1 bind to a common surface on ERH-2, located opposite its PID-3 binding site, explaining how PETISCO can mediate different cellular roles.

Bioaccumulation of therapeutic drugs by human gut bacteria.

Bacteria in the gut can modulate the availability and efficacy of therapeutic drugs. However, the systematic mapping of the interactions between drugs and bacteria has only started recently1 and the main underlying mechanism proposed is the chemical transformation of drugs by microorganisms (biotransformation). Here we investigated the depletion of 15 structurally diverse drugs by 25 representative strains of gut bacteria. This revealed 70 bacteria-drug interactions, 29 of which had not to our knowledge been reported before. Over half of the new interactions can be ascribed to bioaccumulation; that is, bacteria storing the drug intracellularly without chemically modifying it, and in most cases without the growth of the bacteria being affected. As a case in point, we studied the molecular basis of bioaccumulation of the widely used antidepressant duloxetine by using click chemistry, thermal proteome profiling and metabolomics. We find that duloxetine binds to several metabolic enzymes and changes the metabolite secretion of the respective bacteria. When tested in a defined microbial community of accumulators and non-accumulators, duloxetine markedly altered the composition of the community through metabolic cross-feeding. We further validated our findings in an animal model, showing that bioaccumulating bacteria attenuate the behavioural response of Caenorhabditis elegans to duloxetine. Together, our results show that bioaccumulation by gut bacteria may be a common mechanism that alters drug availability and bacterial metabolism, with implications for microbiota composition, pharmacokinetics, side effects and drug responses, probably in an individual manner.

Structural Basis of an Asymmetric Condensin ATPase Cycle.

The condensin protein complex plays a key role in the structural organization of genomes. How the ATPase activity of its SMC subunits drives large-scale changes in chromosome topology has remained unknown. Here we reconstruct, at near-atomic resolution, the sequence of events that take place during the condensin ATPase cycle. We show that ATP binding induces a conformational switch in the Smc4 head domain that releases its hitherto undescribed interaction with the Ycs4 HEAT-repeat subunit and promotes its engagement with the Smc2 head into an asymmetric heterodimer. SMC head dimerization subsequently enables nucleotide binding at the second active site and disengages the Brn1 kleisin subunit from the Smc2 coiled coil to open the condensin ring. These large-scale transitions in the condensin architecture lay out a mechanistic path for its ability to extrude DNA helices into large loop structures.

Upstream of N-Ras C-terminal cold shock domains mediate poly(A) specificity in a novel RNA recognition mode and bind poly(A) binding protein.

RNA binding proteins (RBPs) often engage multiple RNA binding domains (RBDs) to increase target specificity and affinity. However, the complexity of target recognition of multiple RBDs remains largely unexplored. Here we use Upstream of N-Ras (Unr), a multidomain RBP, to demonstrate how multiple RBDs orchestrate target specificity. A crystal structure of the three C-terminal RNA binding cold-shock domains (CSD) of Unr bound to a poly(A) sequence exemplifies how recognition goes beyond the classical ππ-stacking in CSDs. Further structural studies reveal several interaction surfaces between the N-terminal and C-terminal part of Unr with the poly(A)-binding protein (pAbp). All interactions are validated by mutational analyses and the high-resolution structures presented here will guide further studies to understand how both proteins act together in cellular processes.

Structural basis of aggregate binding by the AAA+ disaggregase ClpG.

Severe heat stress causes massive loss of essential proteins by aggregation, necessitating a cellular activity that rescues aggregated proteins. This activity is executed by ATP-dependent, ring-forming, hexameric AAA+ disaggregases. Little is known about the recognition principles of stress-induced protein aggregates. How can disaggregases specifically target aggregated proteins, while avoiding binding to soluble non-native proteins? Here, we determined by NMR spectroscopy the core structure of the aggregate-targeting N1 domain of the bacterial AAA+ disaggregase ClpG, which confers extreme heat resistance to bacteria. N1 harbors a Zn2+-coordination site that is crucial for structural integrity and disaggregase functionality. We found that conserved hydrophobic N1 residues located on a ß-strand are crucial for aggregate targeting and disaggregation activity. Analysis of mixed hexamers consisting of full-length and N1-truncated subunits revealed that a minimal number of four N1 domains must be present in a AAA+ ring for high-disaggregation activity. We suggest that multiple N1 domains increase substrate affinity through avidity effects. These findings define the recognition principle of a protein aggregate by a disaggregase, involving simultaneous contacts with multiple hydrophobic substrate patches located in close vicinity on an aggregate surface. This binding mode ensures selectivity for aggregated proteins while sparing soluble, non-native protein structures from disaggregase activity.

Titin kinase ubiquitination aligns autophagy receptors with mechanical signals in the sarcomere.

Striated muscle undergoes remodelling in response to mechanical and physiological stress, but little is known about the integration of such varied signals in the myofibril. The interaction of the elastic kinase region from sarcomeric titin (A168-M1) with the autophagy receptors Nbr1/p62 and MuRF E3 ubiquitin ligases is well suited to link mechanosensing with the trophic response of the myofibril. To investigate the mechanisms of signal cross-talk at this titin node, we elucidated its 3D structure, analysed its response to stretch using steered molecular dynamics simulations and explored its functional relation to MuRF1 and Nbr1/p62 using cellular assays. We found that MuRF1-mediated ubiquitination of titin kinase promotes its scaffolding of Nbr1/p62 and that the process can be dynamically down-regulated by the mechanical unfolding of a linker sequence joining titin kinase with the MuRF1 receptor site in titin. We propose that titin ubiquitination is sensitive to the mechanical state of the sarcomere, the regulation of sarcomere targeting by Nbr1/p62 being a functional outcome. We conclude that MuRF1/Titin Kinase/Nbr1/p62 constitutes a distinct assembly that predictably promotes sarcomere breakdown in inactive muscle.

Structure and dynamics of the quaternary hunchback mRNA translation repression complex.

A key regulatory process during Drosophila development is the localized suppression of the hunchback mRNA translation at the posterior, which gives rise to a hunchback gradient governing the formation of the anterior-posterior body axis. This suppression is achieved by a concerted action of Brain Tumour (Brat), Pumilio (Pum) and Nanos. Each protein is necessary for proper Drosophila development. The RNA contacts have been elucidated for the proteins individually in several atomic-resolution structures. However, the interplay of all three proteins during RNA suppression remains a long-standing open question. Here, we characterize the quaternary complex of the RNA-binding domains of Brat, Pum and Nanos with hunchback mRNA by combining NMR spectroscopy, SANS/SAXS, XL/MS with MD simulations and ITC assays. The quaternary hunchback mRNA suppression complex comprising the RNA binding domains is flexible with unoccupied nucleotides functioning as a flexible linker between the Brat and Pum-Nanos moieties of the complex. Moreover, the presence of the Pum-HD/Nanos-ZnF complex has no effect on the equilibrium RNA binding affinity of the Brat RNA binding domain. This is in accordance with previous studies, which showed that Brat can suppress mRNA independently and is distributed uniformly throughout the embryo.

Energy-Efficient Decentralized Broadcasting in Wireless Multi-Hop Networks.

Several areas of wireless networking, such as wireless sensor networks or the Internet of Things, require application data to be distributed to multiple receivers in an area beyond the transmission range of a single node. This can be achieved by using the wireless medium's broadcast property when retransmitting data. Due to the energy constraints of typical wireless devices, a broadcasting scheme that consumes as little energy as possible is highly desirable. In this article, we present a novel multi-hop data dissemination protocol called BTP. It uses a game-theoretical model to construct a spanning tree in a decentralized manner to minimize the total energy consumption of a network by minimizing the transmission power of each node. Although BTP is based on a game-theoretical model, it neither requires information exchange between distant nodes nor time synchronization during its operation, and it inhibits graph cycles effectively. The protocol is evaluated in Matlab and NS-3 simulations and through real-world implementation on a testbed of 75 Raspberry Pis. The evaluation conducted shows that our proposed protocol can achieve a total energy reduction of up to 90% compared to a simple broadcast protocol in real-world experiments.

Structure and dynamics of the von Willebrand Factor C6 domain.

Von Willebrand disease (VWD) is a bleeding disorder with different levels of severity. VWD-associated mutations are located in the von Willebrand factor (VWF) gene, coding for the large multidomain plasma protein VWF with essential roles in hemostasis and thrombosis. On the one hand, a variety of mutations in the C-domains of VWF are associated with increased bleeding upon vascular injury. On the other hand, VWF gain-of-function (GOF) mutations in the C4 domain have recently been identified, which induce an increased risk of myocardial infarction. Mechanistic insights into how these mutations affect the molecular behavior of VWF are scarce and holistic approaches are challenging due to the multidomain and multimeric character of this large protein. Here, we determine the structure and dynamics of the C6 domain and the single nucleotide polymorphism (SNP) variant G2705R in C6 by combining nuclear magnetic resonance spectroscopy, molecular dynamics simulations and aggregometry. Our findings indicate that this mutation mostly destabilizes VWF by leading to a more pronounced hinging between both subdomains of C6. Hemostatic parameters of variant G2705R are close to normal under static conditions, but the missense mutation results in a gain-of-function under flow conditions, due to decreased VWF stem stability. Together with the fact that two C4 variants also exhibit GOF characteristics, our data underline the importance of the VWF stem region in VWF's hemostatic activity and the risk of mutation-associated prothrombotic properties in VWF C-domain variants due to altered stem dynamics.

Structure and dynamics of the platelet integrin-binding C4 domain of von Willebrand factor.

Von Willebrand factor (VWF) is a key player in the regulation of hemostasis by promoting recruitment of platelets to sites of vascular injury. An array of 6 C domains forms the dimeric C-terminal VWF stem. Upon shear force activation, the stem adopts an open conformation allowing the adhesion of VWF to platelets and the vessel wall. To understand the underlying molecular mechanism and associated functional perturbations in disease-related variants, knowledge of high-resolution structures and dynamics of C domains is of paramount interest. Here, we present the solution structure of the VWF C4 domain, which binds to the platelet integrin and is therefore crucial for the VWF function. In the structure, we observed 5 intra- and inter-subdomain disulfide bridges, of which 1 is unique in the C4 domain. The structure further revealed an unusually hinged 2-subdomain arrangement. The hinge is confined to a very short segment around V2547 connecting the 2 subdomains. Together with 2 nearby inter-subdomain disulfide bridges, this hinge induces slow conformational changes and positional alternations of both subdomains with respect to each other. Furthermore, the structure demonstrates that a clinical gain-of-function VWF variant (Y2561) is more likely to have an effect on the arrangement of the C4 domain with neighboring domains rather than impairing platelet integrin binding.

DENR-MCTS1 heterodimerization and tRNA recruitment are required for translation reinitiation.

The succession of molecular events leading to eukaryotic translation reinitiation-whereby ribosomes terminate translation of a short open reading frame (ORF), resume scanning, and then translate a second ORF on the same mRNA-is not well understood. Density-regulated reinitiation and release factor (DENR) and multiple copies in T-cell lymphoma-1 (MCTS1) are implicated in promoting translation reinitiation both in vitro in translation extracts and in vivo. We present here the crystal structure of MCTS1 bound to a fragment of DENR. Based on this structure, we identify and experimentally validate that DENR residues Glu42, Tyr43, and Tyr46 are important for MCTS1 binding and that MCTS1 residue Phe104 is important for tRNA binding. Mutation of these residues reveals that DENR-MCTS1 dimerization and tRNA binding are both necessary for DENR and MCTS1 to promote translation reinitiation in human cells. These findings thereby link individual residues of DENR and MCTS1 to specific molecular functions of the complex. Since DENR-MCTS1 can bind tRNA in the absence of the ribosome, this suggests the DENR-MCTS1 complex could recruit tRNA to the ribosome during reinitiation analogously to the eukaryotic initiation factor 2 (eIF2) complex in cap-dependent translation.

Structure, dynamics and roX2-lncRNA binding of tandem double-stranded RNA binding domains dsRBD1,2 of Drosophila helicase Maleless.

Maleless (MLE) is an evolutionary conserved member of the DExH family of helicases in Drosophila. Besides its function in RNA editing and presumably siRNA processing, MLE is best known for its role in remodelling non-coding roX RNA in the context of X chromosome dosage compensation in male flies. MLE and its human orthologue, DHX9 contain two tandem double-stranded RNA binding domains (dsRBDs) located at the N-terminal region. The two dsRBDs are essential for localization of MLE at the X-territory and it is presumed that this involves binding roX secondary structures. However, for dsRBD1 roX RNA binding has so far not been described. Here, we determined the solution NMR structure of dsRBD1 and dsRBD2 of MLE in tandem and investigated its role in double-stranded RNA (dsRNA) binding. Our NMR and SAXS data show that both dsRBDs act as independent structural modules in solution and are canonical, non-sequence-specific dsRBDs featuring non-canonical KKxAXK RNA binding motifs. NMR titrations combined with filter binding experiments and isothermal titration calorimetry (ITC) document the contribution of dsRBD1 to dsRNA binding in vitro. Curiously, dsRBD1 mutants in which dsRNA binding in vitro is strongly compromised do not affect roX2 RNA binding and MLE localization in cells. These data suggest alternative functions for dsRBD1 in vivo.

Muslims' experiences of equality recognition in Germany: Effects on identification with the larger society and on equality recognition of other societal subgroups.

OBJECTIVES: Fostering the identification of societal minorities with the larger society is an essential political aim. In this article, we analyze whether minority members' perception of being recognized by society leads to a stronger identification with society, and whether this identification fosters more willingness to recognize other societal subgroups' members as equal members of society. METHOD: Our analysis is based on both a cross-sectional (N = 1,059, 49% female, mean age = 31 years, 78% with migration background, i.e., first- to third-generation) and a longitudinal (N = 159, 57% female, mean age = 30 years, 76% with migration background) sample of Muslims living in Germany. RESULTS: Structural equation modeling and path analyses suggested that the perception to be recognized by society as equals yielded stronger identification with Germany. In contrast, the perception that one's needs are recognized had no effect on the identification with Germany, while the perception of achievement recognition even tended to decrease identification with Germany. Identification with Germany, in turn, led to an increased willingness of respondents to extend equality recognition to subgroups' members whose beliefs and/or practices our respondents (on average) disapproved. CONCLUSIONS: When the societal majority imparts to minorities a sense of being recognized as equals, this equality recognition colors the broader societal framework within which intergroup relations take shape. We further suggest that-if it is desired that subgroups identify with society-the most viable option is a multicultural model of society, which enables minorities to simultaneously retain their subgroup identity. (PsycINFO Database Record (c) 2020 APA, all rights reserved).

Improving the sensitivity of FT-NMR spectroscopy by apodization weighted sampling.

Apodization weighted acquisition is a simple approach to enhance the sensitivity of multidimensional NMR spectra by scaling the number of scans during acquisition of the indirect dimension(s). The signal content of the resulting spectra is identical to conventionally sampled data, yet the spectra show improved signal-to-noise ratios. There are no special requirements for data acquisition and processing: the time-domain data can be transformed with the same schemes used for conventionally recorded spectra, including Fourier transformation. The method is of general use in multidimensional liquid and solid state NMR experiments if the number of recorded transients per sampling point is bigger than the minimum required phase cycle of the pulse sequence.

Automated structure modeling of large protein assemblies using crosslinks as distance restraints.

Crosslinking mass spectrometry is increasingly used for structural characterization of multisubunit protein complexes. Chemical crosslinking captures conformational heterogeneity, which typically results in conflicting crosslinks that cannot be satisfied in a single model, making detailed modeling a challenging task. Here we introduce an automated modeling method dedicated to large protein assemblies ('XL-MOD' software is available at http://aria.pasteur.fr/supplementary-data/x-links) that (i) uses a form of spatial restraints that realistically reflects the distribution of experimentally observed crosslinked distances; (ii) automatically deals with ambiguous and/or conflicting crosslinks and identifies alternative conformations within a Bayesian framework; and (iii) allows subunit structures to be flexible during conformational sampling. We demonstrate our method by testing it on known structures and available crosslinking data. We also crosslinked and modeled the 17-subunit yeast RNA polymerase III at atomic resolution; the resulting model agrees remarkably well with recently published cryoelectron microscopy structures and provides additional insights into the polymerase structure.

The structure of the box C/D enzyme reveals regulation of RNA methylation.

Post-transcriptional modifications are essential to the cell life cycle, as they affect both pre-ribosomal RNA processing and ribosome assembly. The box C/D ribonucleoprotein enzyme that methylates ribosomal RNA at the 2'-O-ribose uses a multitude of guide RNAs as templates for the recognition of rRNA target sites. Two methylation guide sequences are combined on each guide RNA, the significance of which has remained unclear. Here we use a powerful combination of NMR spectroscopy and small-angle neutron scattering to solve the structure of the 390 kDa archaeal RNP enzyme bound to substrate RNA. We show that the two methylation guide sequences are located in different environments in the complex and that the methylation of physiological substrates targeted by the same guide RNA occurs sequentially. This structure provides a means for differential control of methylation levels at the two sites and at the same time offers an unexpected regulatory mechanism for rRNA folding.

The structure of the SOLE element of oskar mRNA.

mRNA localization by active transport is a regulated process that requires association of mRNPs with protein motors for transport along either the microtubule or the actin cytoskeleton. oskar mRNA localization at the posterior pole of the Drosophila oocyte requires a specific mRNA sequence, termed the SOLE, which comprises nucleotides of both exon 1 and exon 2 and is assembled upon splicing. The SOLE folds into a stem-loop structure. Both SOLE RNA and the exon junction complex (EJC) are required for oskar mRNA transport along the microtubules by kinesin. The SOLE RNA likely constitutes a recognition element for a yet unknown protein, which either belongs to the EJC or functions as a bridge between the EJC and the mRNA. Here, we determine the solution structure of the SOLE RNA by Nuclear Magnetic Resonance spectroscopy. We show that the SOLE forms a continuous helical structure, including a few noncanonical base pairs, capped by a pentanucleotide loop. The helix displays a widened major groove, which could accommodate a protein partner. In addition, the apical helical segment undergoes complex dynamics, with potential functional significance.

Multi-domain conformational selection underlies pre-mRNA splicing regulation by U2AF.

Many cellular functions involve multi-domain proteins, which are composed of structurally independent modules connected by flexible linkers. Although it is often well understood how a given domain recognizes a cognate oligonucleotide or peptide motif, the dynamic interaction of multiple domains in the recognition of these ligands remains to be characterized. Here we have studied the molecular mechanisms of the recognition of the 3'-splice-site-associated polypyrimidine tract RNA by the large subunit of the human U2 snRNP auxiliary factor (U2AF65) as a key early step in pre-mRNA splicing. We show that the tandem RNA recognition motif domains of U2AF65 adopt two remarkably distinct domain arrangements in the absence or presence of a strong (that is, high affinity) polypyrimidine tract. Recognition of sequence variations in the polypyrimidine tract RNA involves a population shift between these closed and open conformations. The equilibrium between the two conformations functions as a molecular rheostat that quantitatively correlates the natural variations in polypyrimidine tract nucleotide composition, length and functional strength to the efficiency to recruit U2 snRNP to the intron during spliceosome assembly. Mutations that shift the conformational equilibrium without directly affecting RNA binding modulate splicing activity accordingly. Similar mechanisms of cooperative multi-domain conformational selection may operate more generally in the recognition of degenerate nucleotide or amino acid motifs by multi-domain proteins.

Segmental, Domain-Selective Perdeuteration and Small-Angle Neutron Scattering for Structural Analysis of Multi-Domain Proteins.

Multi-domain proteins play critical roles in fine-tuning essential processes in cellular signaling and gene regulation. Typically, multiple globular domains that are connected by flexible linkers undergo dynamic rearrangements upon binding to protein, DNA or RNA ligands. RNA binding proteins (RBPs) represent an important class of multi-domain proteins, which regulate gene expression by recognizing linear or structured RNA sequence motifs. Here, we employ segmental perdeuteration of the three RNA recognition motif (RRM) domains in the RBP TIA-1 using Sortase A mediated protein ligation. We show that domain-selective perdeuteration combined with contrast-matched small-angle neutron scattering (SANS), SAXS and computational modeling provides valuable information to precisely define relative domain arrangements. The approach is generally applicable to study conformational arrangements of individual domains in multi-domain proteins and changes induced by ligand binding.

Synthesis of Highly Selective Submicromolar Microcystin-Based Inhibitors of Protein Phosphatase (PP)2A over PP1.

Research and therapeutic targeting of the phosphoserine/threonine phosphatases PP1 and PP2A is hindered by the lack of selective inhibitors. The microcystin (MC) natural toxins target both phosphatases with equal potency, and their complex synthesis has complicated structure-activity relationship studies in the past. We report herein the synthesis and biochemical evaluation of 11 MC analogues, which was accomplished through an efficient strategy combining solid- and solution-phase approaches. Our approach led to the first MC analogue with submicromolar inhibitory potency that is strongly selective for PP2A over PP1 and does not require the complex lipophilic Adda group. Through mutational and structural analyses, we identified a new key element for binding, as well as reasons for the selectivity. This work gives unprecedented insight into how selectivity between these phosphatases can be achieved with MC analogues.