Intron-containing mRNAs are cotranscriptionally spliced and assembled into messenger ribonucleoprotein (mRNP) particles, a process monitored by surveillance pathways. Here, we combined biochemical and structural approaches to elucidate the mechanisms by which mRNPs are sorted between two opposing fates: nuclear degradation and cytoplasmic export. While the human GANP-PCID2 complex is known to connect mRNPs to nuclear export, our data indicate that the LENG8-PCID2 complex operates as an mRNP decay connector, coupling nuclear mRNPs to the RNA-degrading exosome via the PAXT adaptor complex. Both recognize the mRNP component UAP56, but LENG8-PCID2 uniquely associates with early splicing factors through a direct interaction with U1A and RRP1B. Similarly, the Thp3-Csn12 ortholog in budding yeast couples the early splicing factors Mud2-Bbp with the nuclear exosome. The spliceosome-exosome mRNP decay pathway we uncovered reveals molecular principles that remain strikingly conserved across evolution, despite the fundamental differences in splicing and decay between humans and budding yeast.

Upf1 is a master regulator of nonsense-mediated mRNA decay (NMD), an mRNA surveillance and degradation pathway conserved from yeast to human. In Saccharomyces cerevisiae, Upf1 exists in two distinct complexes with factors that mediate NMD activation or 5'-3' mRNA degradation. We combined endogenous purifications and biochemical reconstitutions of yeast Upf1 complexes with structural analyses and biochemical assays to elucidate the molecular mechanisms driving the organization of the Upf1-5'-3' and Upf1-2-3 complexes. We show that yeast Upf1 is in a constitutive complex, whereby its CH, RecA, and C-terminal domains interact with the mRNA decapping factor Dcp2, NMD-associated proteins Nmd4 and Ebs1, and the 5'-3' exoribonuclease Xrn1, respectively. Together, the interacting surfaces and closed conformation of Upf1 in the Upf1-5'-3' complex sterically obstruct the binding of Upf2-3. Our work points to a major restructuring upon recruitment of these factors during NMD and provides insights into evolutionary divergence amongst species.

The lifespan of most eukaryotic mRNAs is modulated by the gradual shortening of the poly(A) tail and removal of the associated poly(A)-binding protein. The human PAN2-PAN3 complex catalyzes initial deadenylation by shortening long poly(A) tails associated with PABPC1. Both PAN2-PAN3 and PABPC1 are evolutionarily conserved from fungi to humans. How the human complex has adapted to recognize and act on longer poly(A) tails characteristic of mammalian mRNAs remains unclear. Here, we report a method to obtain homo-polymeric poly(A) RNAs up to 240 nt, mimicking the synthesis length of poly(A) tails in mammals. We recapitulate human deadenylation properties in vitro, with PAN2-PAN3 showing greater activity on long poly(A)-PABPC1 ribonucleoprotein substrates. Single-particle cryo-electron microscopy (cryo-EM) analyses of PAN2-PAN3 bound to poly(A)-PABPC1 ribonucleoproteins uncover a longer substrate-binding path in the case of the human deadenylase compared to fungi. Altogether, these data provide a rationale for the co-evolution of deadenylase properties and poly(A) tail lengths.

Background Cluster of differentiation 95 (CD95/Fas/Apo1) as part of the Tumor-necrosis factor (TNF) receptor family is a prototypic trigger of the ‘extrinsic’ apoptotic pathway and its activation by the trimeric ligand CD95L is of high interest. However, CD95L, when presented in solution, exhibits a low efficiency to induce apoptosis signaling in human cells. Results Here, we design a recombinant CD95L exhibiting an isoleucine zipper (IZ) motif at the N-terminus for stabilization of the trimerized CD95L and demonstrate its high apoptosis initiation efficiency. This efficiency is further enhanced by antibody-mediated crosslinking of IZ-CD95L.A cysteine amino acid fused behind the IZ is used as a versatile coupling site for bionanotechnological applications or for the development of biomedical assays. A fast, cheap, and efficient production of CD95L via the HEK293T secretory expression system is presented, along with CD95L affinity purification and functionalization. We verified the biological activity of the purified protein and identified a stabilized trimeric CD95L structure as the most potent inducer of apoptosis signaling. Conclusions The workflow and the findings reported here will streamline a wide array of future low- or high-throughput TNF-ligand screens, and their modification towards improving apoptosis induction efficiency and, potentially, anticancer therapy.

Background: Cluster of differentiation 95 (CD95/Fas/Apo1) as part of the Tumor-necrosis factor (TNF) receptor family is a prototypic trigger of the extrinsic apoptotic pathway and its activation by the trimeric ligand CD95L is of high interest for anticancer therapy. However, CD95L, when presented in solution, exhibits a low efficiency to induce apoptosis signaling in human cells. Results: Here, we design a recombinant CD95L exhibiting an isoleucine zipper (IZ) motif at the N-terminus for stabilization of the trimerized CD95L and demonstrate its high apoptosis induction efficiency. A cysteine amino acid fused behind the IZ is further used as a versatile coupling site for bionanotechnological applications or for the development of biomedical assays. A fast, cheap, and high-yield production of CD95L via the HEK293T secretory expression system is presented, along with CD95L affinity purification and functionalization. We verified the biological activity of the purified protein and identified a stabilized trimeric CD95L structure as the most potent inducer of apoptosis signaling. Conclusions: The workflow and the findings reported here will streamline a wide array of future low- or high-throughput TNF-ligand screens, and their modification towards improving apoptosis induction efficiency and anticancer therapy. ### Competing Interest Statement The authors have declared no competing interest.

Cyclic nucleotide binding domains (CNB) confer allosteric regulation by cAMP or cGMP to many signaling proteins, including PKA and PKG. PKA of phylogenetically distant Trypanosoma is the first exception as it is cyclic nucleotide-independent and responsive to nucleoside analogues (Bachmaier et al., 2019). Here, we show that natural nucleosides inosine, guanosine and adenosine are nanomolar affinity CNB ligands and activators of PKA orthologs of the important tropical pathogens Trypanosoma brucei, Trypanosoma cruzi, and Leishmania. The sequence and structural determinants of binding affinity, -specificity and kinase activation of PKAR were established by structure-activity relationship (SAR) analysis, co-crystal structures and mutagenesis. Substitution of two to three amino acids in the binding sites is sufficient for conversion of CNB domains from nucleoside to cyclic nucleotide specificity. In addition, a trypanosomatid-specific C-terminal helix (αD) is required for high affinity binding to CNB-B. The αD helix functions as a lid of the binding site that shields ligands from solvent. Selectivity of guanosine for CNB-B and of adenosine for CNB-A results in synergistic kinase activation at low nanomolar concentration. PKA pulldown from rapid lysis establishes guanosine as the predominant ligand in vivo in T. brucei bloodstream forms, whereas guanosine and adenosine seem to synergize in the procyclic developmental stage in the insect vector. We discuss the versatile use of CNB domains in evolution and recruitment of PKA for novel nucleoside-mediated signaling.

Gene expression during natural and induced reprogramming is controlled by pioneer transcription factors that initiate transcription from closed chromatin. Nr5a2 is a key pioneer factor that regulates zygotic genome activation in totipotent embryos, pluripotency in embryonic stem cells and metabolism in adult tissues, but the mechanism of its pioneer activity remains poorly understood. Here, we present a cryo-electron microscopy structure of human NR5A2 bound to a nucleosome. The structure shows that the conserved carboxy-terminal extension (CTE) loop of the NR5A2 DNA-binding domain competes with a DNA minor groove anchor of the nucleosome and releases entry-exit site DNA. Mutational analysis showed that NR5A2 D159 of the CTE is dispensable for DNA binding but required for stable nucleosome association and persistent DNA ‘unwrapping’. These findings suggest that NR5A2 belongs to an emerging class of pioneer factors that can use DNA minor groove anchor competition to destabilize nucleosomes and facilitate gene expression during reprogramming.

Nonsense-mediated mRNA decay (NMD) is a conserved co-translational mRNA surveillance and turnover pathway across eukaryotes. NMD has a central role in degrading defective mRNAs and also regulates the stability of a significant portion of the transcriptome. The pathway is organized around UPF1, an RNA helicase that can interact with several NMD-specific factors. In human cells, degradation of the targeted mRNAs begins with a cleavage event that requires the recruitment of the SMG6 endonuclease to UPF1. Previous studies have identified functional links between SMG6 and UPF1, but the underlying molecular mechanisms have remained elusive. Here, we used mass spectrometry, structural biology and biochemical approaches to identify and characterize a conserved short linear motif in SMG6 that interacts with the cysteine/histidine-rich (CH) domain of UPF1. Unexpectedly, we found that the UPF1-SMG6 interaction is precluded when the UPF1 CH domain is engaged with another NMD factor, UPF2. Based on cryo-EM data, we propose that the formation of distinct SMG6-containing and UPF2-containing NMD complexes may be dictated by different conformational states connected to the RNA-binding status of UPF1. Our findings rationalize a key event in metazoan NMD and advance our understanding of mechanisms regulating activity and guiding substrate recognition by the SMG6 endonuclease.

Engineering of biosynthetic enzymes is increasingly employed to synthesize structural analogues of antibiotics. Of special interest are nonribosomal peptide synthetases (NRPSs) responsible for the production of important antimicrobial peptides. Here, directed evolution of an adenylation domain of a Pro-specific NRPS module completely switched substrate specificity to the non-standard amino acid piperazic acid (Piz) bearing a labile N-N bond. This success was achieved by UPLC-MS/MS-based screening of small, rationally designed mutant libraries and can presumably be replicated with a larger number of substrates and NRPS modules. The evolved NRPS produces a Piz-derived gramicidin S analogue. Thus, we give new impetus to the too-early dismissed idea that widely accessible low-throughput methods can switch the specificity of NRPSs in a biosynthetically useful fashion.

Mammalian macrophage migration inhibitory factor (MIF) and its paralog, D-dopachrome tautomerase, are multifunctional inflammatory cytokines. Plants have orthologous MIF and D-dopachrome tautomerase–like (MDL) proteins that mimic some of the effects of MIF on immune cells in vitro. We explored the structural and functional similarities between the three Arabidopsis thaliana MDLs and MIF. X-ray crystallography of the MDLs revealed high structural similarity between MDL and MIF homotrimers and suggested a potential explanation for the lack of tautomerase activity in the MDLs. MDL1 and MDL2 interacted with each other and with MIF in vitro, in yeast, and in plant leaves and formed hetero-oligomeric complexes with MIF in vitro. The MDLs stimulated signaling through the MIF receptors CXCR2 or CXCR4 and enhanced the responses to MIF in a yeast reporter system, in human neutrophils, and in human lung epithelial cells. Pharmacological inhibitors that disrupted MIF activity or prevented the formation of MIF-MDL hetero-oligomers blocked the observed synergism. These findings demonstrate that MDLs can enhance cellular responses to MIF, which may have functional implications in tissues exposed to MDLs from the diet or environment.

The evolutionary conserved CCR4-NOT complex functions in the cytoplasm as the main mRNA deadenylase in both constitutive mRNA turnover and regulated mRNA decay pathways. The versatility of this complex is underpinned by its modular multi-subunit organization, with distinct structural modules actuating different functions. The structure and function of all modules are known, except for that of the N-terminal module. Using different structural approaches, we obtained high-resolution data revealing the architecture of the human N-terminal module composed of CNOT1, CNOT10, and CNOT11. The structure shows how two helical domains of CNOT1 sandwich CNOT10 and CNOT11, leaving the most conserved domain of CNOT11 protruding into solvent as an antenna. We discovered that GGNBP2, a protein identified as a tumor suppressor and spermatogenic factor, is a conserved interacting partner of the CNOT11 antenna domain. Structural and biochemical analyses thus pinpoint the N-terminal CNOT1-CNOT10-CNOT11 module as a conserved protein-protein interaction platform.

Messenger RNAs (mRNAs) are at the center of the central dogma of molecular biology. In eukaryotic cells, these long ribonucleic acid polymers do not exist as naked transcripts; rather, they associate with mRNA-binding proteins to form messenger ribonucleoprotein (mRNP) complexes. Recently, global proteomic and transcriptomic studies have provided comprehensive inventories of mRNP components. However, knowledge of the molecular features of distinct mRNP populations has remained elusive. We purified endogenous nuclear mRNPs fromSaccharomyces cerevisiaeby harnessing the mRNP biogenesis factors THO and Sub2 in biochemical procedures optimized to preserve the integrity of these transient ribonucleoprotein assemblies. We found that these mRNPs are compact particles that contain multiple copies of Yra1, an essential protein with RNA-annealing properties. To investigate their molecular and architectural organization, we used a combination of proteomics, RNA sequencing, cryo-electron microscopy, cross-linking mass spectrometry, structural models, and biochemical assays. Our findings indicate that yeast nuclear mRNPs are packaged around an intricate network of interconnected proteins capable of promoting RNA–RNA interactions via their positively charged intrinsically disordered regions. The evolutionary conservation of the major mRNA-packaging factor (yeast Yra1 and Aly/REF in metazoans) points toward a general paradigm governing nuclear mRNP packaging.

Calcium in interstitial fluids is central to systemic physiology and a crucial ion pool for entry into cells through numerous plasma membrane channels. Its study has been limited by the scarcity of methods that allow monitoring in tight inter-cell spaces of living tissues. Here we present high performance ultra-low affinity genetically encoded calcium biosensors named GreenT-ECs. GreenT-ECs combine large fluorescence changes upon calcium binding and binding affinities (Kds) ranging from 0.8 mM to 2.9 mM, making them tuned to calcium concentrations in extracellular organismal fluids. We validated GreenT-ECs in rodent hippocampal neurons and transgenic zebrafish in vivo, where the sensors enabled monitoring homeostatic regulation of tissue interstitial calcium. GreenT-ECs may become useful for recording very large calcium transients and for imaging calcium homeostasis in inter-cell structures in live tissues and organisms.

AbstractDas Engineering biosynthetischer Enzyme wird zunehmend eingesetzt, um strukturelle Analoga von Antibiotika zu synthetisieren. Von besonderem Interesse sind nichtribosomale Peptidsynthetasen (NRPS), die für die Produktion wichtiger antimikrobieller Peptide verantwortlich sind. Hier beschreiben wir, wie durch gerichtete Evolution einer Adenylierungsdomäne eines Pro‐spezifischen NRPS‐Moduls dessen Substratspezifität vollständig auf die Nicht‐Standard‐Aminosäure Piperazinsäure (Piz) geändert wurde, welche eine labile N−N‐Bindung enthält. Dieser Erfolg wurde durch UPLC‐MS/MS‐basiertes Screening kleiner, rational konzipierter Mutantenbibliotheken erzielt und kann vermutlich mit einer größeren Anzahl von Substraten und NRPS‐Modulen repliziert werden. Die evolvierte NRPS ist in der Lage, ein Piz‐beinhaltendes Gramicidin S‐Analogon zu produzieren. Damit geben wir der zu früh verworfenen Idee neuen Auftrieb, dass weithin zugängliche Methoden mit niedrigem Durchsatz die Spezifität von NRPS in einer biosynthetisch nützlichen Weise verändern können.

Vertebrates have developed effective immune mechanisms to fight microbial attacks, relying on a sophisticated network of innate and adaptive responses, a circulatory system, and numerous orchestrating soluble mediators such as cytokines. Mammalian macrophage migration inhibitory factor (MIF) and its paralog D-dopachrome tautomerase (D-DT/MIF-2) are multifunctional inflammatory cytokines with chemokine-like properties that modulate immunity. Plants possess orthologous MIF/D-DT-like (MDL) proteins, whose function is largely unexplored. Driven by the previous discovery of cross-kingdom mimicry of plant ( Arabidopsis thaliana ) MDL proteins and human MIF receptor signaling, we here characterized the structures of the three A. thaliana MDLs by X-ray crystallography and explored the mechanism underlying the interplay between plant MDLs, human MIF, and its receptors. We obtained high-resolution structures at 1.56 Å, 1.40 Å, and 2.00 Å resolution for MDL1, MDL2, and MDL3, respectively, revealing a typical trimeric assembly and a high three-dimensional similarity to human MIF. Although residues at the catalytic site of the three MDLs show high identity to human MIF, the proteins showed low tautomerase activity for the substrate 4-hydroxyphenylpyruvate (HPP). Structural differences likely explain the enzymatic inactivity of plant MDLs for HPP. Strikingly, employing in vitro, in vivo, and in planta test systems, we found that MIF and MDL proteins interact with each other and have the capacity to form hetero-oligomeric complexes. The functional consequences of this interaction were demonstrated applying a yeast-based reporter system specific for the MIF chemokine receptors CXCR2 and CXCR4. MDLs not only triggered receptor signaling on their own, but exhibited pronounced synergism regarding the activation of the CXCR2- and CXCR4-dependent signaling pathways, when co-applied with MIF. These findings were substantiated by the co-administration of pharmacological inhibitors that either disrupt MIF receptor binding or block the catalytic cavity. Moreover, biochemical and biophysical experiments using an allosteric oligomer-specific MIF inhibitor established hexa-oligomer formation between MIF and MDLs as the putative basis for the synergistic effect. Our results are the starting point for a mechanistic understanding of the immunomodulatory activity of a family of highly conserved plant proteins. One Sentence Summary A. thaliana MDLs and human macrophage inhibitory factor interact with each other and induce synergistic effects in activating CXCR2 and CXCR4.

Fun30 is the prototype of the Fun30-SMARCAD1-ETL subfamily of nucleosome remodelers involved in DNA repair and gene silencing. These proteins appear to act as single-subunit nucleosome remodelers, but their molecular mechanisms are, at this point, poorly understood. Using multiple sequence alignment and structure prediction, we identify an evolutionarily conserved domain that is modeled to contain a SAM-like fold with one long, protruding helix, which we term SAM-key. Deletion of the SAM-key within budding yeast Fun30 leads to a defect in DNA repair and gene silencing similar to that of the fun30Δ mutant. In vitro, Fun30 protein lacking the SAM-key is able to bind nucleosomes but is deficient in DNA-stimulated ATPase activity and nucleosome sliding and eviction. A structural model based on AlphaFold2 prediction and verified by crosslinking-MS indicates an interaction of the long SAM-key helix with protrusion I, a subdomain located between the two ATPase lobes that is critical for control of enzymatic activity. Mutation of the interaction interface phenocopies the domain deletion with a lack of DNA-stimulated ATPase activation and a nucleosome-remodeling defect, thereby confirming a role of the SAM-key helix in regulating ATPase activity. Our data thereby demonstrate a central role of the SAM-key domain in mediating the activation of Fun30 catalytic activity, thus highlighting the importance of allosteric activation for this class of enzymes.

The coronavirus disease 2019 (COVID-19) pandemic is fading, however its etiologic agent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues posing - despite the availability of licensed vaccines – a global health threat, due to the potential emergence of vaccine-resistant SARS-CoV-2 variants. This makes the development of new drugs against COVID-19 a persistent urgency and sets as research priority the validation of novel therapeutic targets within the SARS-CoV-2 proteome. Among these, a promising one is the SARS-CoV-2 nucleocapsid (N) phosphoprotein, a major structural component of the virion with indispensable role in packaging the viral genome into a ribonucleoprotein (RNP) complex, which also contributes to SARS-CoV-2 innate immune evasion by inhibiting the host cell type-I interferon (IFN-I) response. By combining miniaturized differential scanning fluorimetry with microscale thermophoresis, we found that the 100-year-old drug Suramin interacts with SARS-CoV-2 N-terminal domain (NTD) and C-terminal domain (CTD), thereby inhibiting their single-stranded RNA (ssRNA) binding function with low-micromolar Kd and IC50 values. Molecular docking suggests that Suramin interacts with basic NTD cleft and CTD dimer interface groove, highlighting three potentially druggable ssRNA binding sites. Electron microscopy shows that Suramin inhibits the formation in vitro of RNP complex-like condensates by SARS-CoV-2 N with a synthetic ssRNA. In a dose-dependent manner, Suramin also reduced SARS-CoV-2-induced cytopathic effect on Vero E6 and Calu-3 cells, partially reverting the SARS-CoV-2 N-inhibited IFN-I production in 293T cells. Our findings indicate that Suramin inhibits SARS-CoV-2 replication by hampering viral genome packaging, thereby representing a starting model for design of new COVID-19 antivirals.

Most proteins fold into 3D structures that determine how they function and orchestrate the biological processes of the cell. Recent developments in computational methods for protein structure predictions have reached the accuracy of experimentally determined models. Although this has been independently verified, the implementation of these methods across structural-biology applications remains to be tested. Here, we evaluate the use of AlphaFold2 (AF2) predictions in the study of characteristic structural elements; the impact of missense variants; function and ligand binding site predictions; modeling of interactions; and modeling of experimental structural data. For 11 proteomes, an average of 25% additional residues can be confidently modeled when compared with homology modeling, identifying structural features rarely seen in the Protein Data Bank. AF2-based predictions of protein disorder and complexes surpass dedicated tools, and AF2 models can be used across diverse applications equally well compared with experimentally determined structures, when the confidence metrics are critically considered. In summary, we find that these advances are likely to have a transformative impact in structural biology and broader life-science research.

AbstractMedium‐chain alcohol dehydrogenases (ADHs) comprise a highly conserved enzyme family that catalyse the reversible reduction of aldehydes. However, recent discoveries in plant natural product biosynthesis suggest that the catalytic repertoire of ADHs has been expanded. Here we report the crystal structure of dihydroprecondylocarpine acetate synthase (DPAS), an ADH that catalyses the non‐canonical 1,4‐reduction of an α,β‐unsaturated iminium moiety. Comparison with structures of plant‐derived ADHs suggest the 1,4‐iminium reduction does not require a proton relay or the presence of a catalytic zinc ion in contrast to canonical 1,2‐aldehyde reducing ADHs that require the catalytic zinc and a proton relay. Furthermore, ADHs that catalysed 1,2‐iminium reduction required the presence of the catalytic zinc and the loss of the proton relay. This suggests how the ADH active site can be modified to perform atypical carbonyl reductions, providing insight into how chemical reactions are diversified in plant metabolism.

Thousands of natural products are derived from the fused cyclopentane-pyran molecular scaffold nepetalactol. These natural products are used in an enormous range of applications that span the agricultural and medical industries. For example, nepetalactone, the oxidized derivative of nepetalactol, is known for its cat attractant properties as well as potential as an insect repellent. Most of these naturally occurring nepetalactol-derived compounds arise from only two out of the eight possible stereoisomers, 7 S - cis - trans and 7 R - cis - cis nepetalactols. Here we use a combination of naturally occurring and engineered enzymes to produce seven of the eight possible nepetalactol or nepetalactone stereoisomers. These enzymes open the possibilities for biocatalytic production of a broader range of iridoids, providing a versatile system for the diversification of this important natural product scaffold.