Nature Communications

Here, we report the discovery of a ParM protein from Clostridium botulinum (CBgs-ParM), which forms a double-stranded polar filament. CBgs-ParM shares many similarities in its basic filament architecture with actin, however, Pi release after nucleotide hydrolysis induces a large lateral strand shift of ~2.5 nm. We identified the ParR (CBgs-ParR) that acts as a nucleation factor in the initial stage of polymerization, similar to ParR from Escherichia coli plasmid R1. CBgs-ParR also functions as a depolymerization factor, probably by recognizing the structural change in the CBgs-ParM filament after Pi release. Comparison with CBH-ParM, another ParM from Clostridum botulinum, showed that subunit-subunit interacting regions largely differ, preventing co-polymerization, implying a selection pressure in evolution to prevent interference between different ParMRC systems.

Mammalian Ca2+-dependent Slo K+ channels are expressed with {beta} and {gamma} auxiliary subunits that greatly influence voltage- and Ca2+-induced gating, thereby fundamentally altering the behavior of the channel. The four {gamma} subunits reduce the need for voltage-dependent activation, allowing Slo to open in the absence of an action potential. The mechanism of this activation has, however, remained elusive. Here, we present the cryo-EM structure of Slo1 in complex with {gamma}1/LRRC26, revealing how the transmembrane helix of {gamma}1 binds and presumably stabilizes the active conformation of the voltage-sensor domain. This effect is further enhanced by a polybasic stretch on the intracellular side of the membrane which locally changes the charge gradient across the membrane. Sequence differences explain why the four {gamma} subunits possess different activation efficiencies. Simultaneous binding of {gamma} and the unrelated {beta} subunits is structurally possible, as both binding sites do not overlap and the {gamma}1 LRR domains are partially flexible. Thus, our data provide a possible explanation for Slo1 regulation by {gamma} subunits, and furthermore suggest a novel mechanism of activation of voltage-gated ion channels by auxiliary subunits and add to the growing knowledge of their complex regulation.

Cellulosomes are efficient enzymatic nanomachines which have arisen for the degradation of cellulosic biomass. They are found abundantly in soil-dwelling microbes and bacteria which thrive in the stomachs of ruminant mammals. Two protein domains, cohesins and dockerins, characterise cellulosomes. These domains interact with each other to form enormous complexes with as many as 130 individual proteins. Annotation of the genome of Cellulosilyticum lenotcellum DSM 5427 revealed one single cohesin and one single dockerin domain. This pales in comparison to most cellulosomal organisms. We have recombinantly produced the proteins which bear these domains and demonstrated cellulase activity, which is enhanced by interaction between the two proteins. Moreover we have identified additional novel interacting partners for this unique cellulosome complex. This broadens the repertoire and definitions of cellulosomes overall.

In plant mitochondria and chloroplasts, cytidine-to-uridine RNA editing plays a crucial role in regulating gene expression. While natural PLS-type PPR proteins are specialized in this process, synthetic PPR proteins offer significant potential for targeted RNA editing. In this study, we engineered chimeric editing factors by fusing synthetic P-type PPR guides with the DYW cytidine deaminase domain of a moss mitochondrial editing factor, PPR56. These designer PPR editors (dPPRe) elicited efficient and precise de novo RNA editing in Escherichia coli, and in Nicotiana benthamiana chloroplasts and mitochondria. Chloroplast transcriptome-wide analysis of the most efficient dPPRe revealed minimal off-target effects, with only three non-target C sites edited due to sequence similarity with the intended target. This study introduces a novel and precise method for RNA base editing in plant organelles, paving the way for new approaches in gene regulation applicable to plants and potentially other organisms.

In Archaea and Eukaryotes, the synthesis of a universal tRNA modification, t6A, is catalyzed by the KEOPS complex composed of Kae1, Bud32, Cgi121 and Pcc1. A fifth subunit, Gon7, is found only in Fungi and Metazoa. Mutations in all five genes encoding human KEOPS subunits leads to Galloway-Mowat syndrome, a severe genetic disease causing childhood lethality. Here, we describe the discovery and biochemical characterization of the archaeal fifth KEOPS subunit. This protein, dubbed Pcc2, is a paralog of Pcc1 and is widely conserved in Archaea. Pcc1 and Pcc2 form a heterodimer in solution, show modest sequence conservation but very high structural similarity. The 5-subunit KEOPS lost its capacity to form dimers but its tRNA binding and t6A synthetic activity remained robust. Pcc2 can substitute Pcc1 but the resulting KEOPS complex is inactive suggesting a distinct function for the two paralogs. Comparative sequence and structure analyses point to a possible evolutionary link between archaeal Pcc2 and eukaryotic Gon7 proteins. Our work thus reveals that Pcc2 has evolved to regulate the oligomeric state of KEOPS complex thus adding another layer of complexity to the biosynthesis of t6A that seems to be conserved from Archaea to Eukaryotes.

Uric acid is the main means of nitrogen excretion in uricotelic vertebrates (birds and reptiles) and the end product of purine catabolism in humans and a few other mammals. While uricase is inactivated in mammals unable to degrade urate, the presence of orthologous genes without inactivating mutations in avian and reptilian genomes is unexplained. Here we show that the Gallus gallus gene we name cysteine-rich urate oxidase (CRUOX) encodes a functional protein representing a unique case of cysteine enrichment in the evolution of vertebrate orthologous genes. CRUOX retains the ability to catalyze urate oxidation to hydrogen peroxide and 5-hydroxyisourate (HIU), albeit with a 100-fold reduced efficiency. However, differently from all uricases hitherto characterized, it can also facilitate urate regeneration from HIU, a catalytic property which we propose depends on its enrichment in cysteine residues. X-ray structural analysis highlights differences in the active site compared to known orthologs and suggests a mechanism for cysteine-mediated self-aggregation under H2O2-oxidative conditions. Cysteine enrichment was concurrent with transition to uricotelism and a shift in gene expression from the liver to the skin where CRUOX is co-expressed with {beta}-keratins. Therefore, the loss of urate degradation in amniotes has followed opposite evolutionary trajectories: while uricase has been eliminated by pseudogenization in some mammals, it has been repurposed as a redox-sensitive enzyme in the reptilian skin.

We could demonstrate that the AAA+ unfoldase ClpC together with the protein arginine kinase and adaptor protein McsB, its activator McsA and the phosphatase YwlE form a unique chaperone system. Here, the McsA-activated McsB phosphorylates and targets aggregated substrate proteins for extraction and unfolding by ClpC. Sub-stoichiometric amounts of the YwlE phosphatase enhanced the ClpC/McsB/McsA mediated disaggregation and facilitated the de-phosphorylation of the arginine-phosphorylated substrate protein extruded by ClpC, allowing its subsequent refolding. Interestingly, the successfully refolded protein escaped degradation by the loosely associated ClpP protease. This unique chaperone system is thereby able to disaggregate and refold aggregated proteins but can also remove severely damaged protein aggregates by degradation.

The rise of antifungal resistance is a global challenge for both human health and food security, because resistance emergence easily outpaces the antifungal development pipeline. Furthermore, resistance arises often in parallel and through alternative mechanisms creating challenges to predict emergence. In agriculture, where vast areas are sprayed by diverse cocktails, antifungal resistance gains are particularly complex. Despite broad efforts, knowledge of resistance mechanisms is often limited to model genotypes and empirical evidence from the field is lacking. Here, we define and validate a high- throughput pipeline for antifungal resistance discovery informed by emerging resistance gains at continental scale. We analyzed a thousand-genome European diversity panel of the major wheat pathogen Zymoseptoria tritici and assessed resistance levels against over 29 fungicides covering all major classes. We optimized high-throughput phenotyping assays to comprehensively capture emerging resistance phenotypes. Pangenome-informed genotyping techniques revealed a total of 2192 genes associated with antifungal resistance. This expands by an order of magnitude the current knowledge and establishes a refined atlas of resistance mutations. We generated mutants to recapitulate several of the discovered resistance factors. Hence, our approach captures in-field resistance gains across Europe for all major fungicide classes and can define exact molecular targets. Broad knowledge of resistance gains will guide more sustainable fungicide development pipelines.

Here we describe the cryo-electron microscopy structure of the human histamine 2 receptor (H2R) in an active conformation with bound histamine and in complex with Gs heterotrimeric protein at an overall resolution of 3.4 [A]. The complex was generated by cotranslational insertion into preformed nanodisc membranes using cell-free synthesis in E. coli lysates. It is the first structure obtained by this detergent-free strategy and the first GPCR/Gs complex structure in lipid environment. Structural comparison with the inactive conformation of H2R and the inactive and Gq-coupled active state of H1R together with structure-guided functional experiments reveal molecular insights into the specificity of ligand binding and G protein coupling for this receptor family. We demonstrate lipid-modulated folding of cell-free synthesized H2R, its agonist-dependent internalization and its interaction with endogenously synthesized H1R and H2R in HEK293 cells by applying a recently developed nanotransfer technique.

Homing-based gene drives are novel interventions promising the area-wide, species-specific genetic control of harmful insect populations. Here we characterise a first set of gene drives in a tephritid agricultural pest species, the Mediterranean fruit fly Ceratitis capitata (medfly). Our results show that the medfly is highly amenable to homing-based gene drive strategies. By targeting the medfly transformer gene, we also demonstrate two different mechanisms by which CRISPR-Cas9 gene drive can be coupled to sex conversion, whereby genetic females are transformed into fertile and harmless XX males. Given this unique malleability of sex determination, we modelled gene drive interventions that couple sex conversion and female sterility and found that such approaches could be effective and tolerant of resistant allele selection in the target population. Our results open the door for developing gene drive strains for the population suppression of the medfly and related tephritid pests by co-targeting female reproduction and shifting the reproductive sex ratio towards males. They demonstrate the untapped potential for gene drives to tackle agricultural pests in an environmentally friendly and economical way.

RepA is a bacterial protein that builds intracellular amyloid oligomers acting as inhibitory complexes of plasmid DNA replication. When carrying a mutation enhancing its amyloidogenesis (A31V), the N-terminal domain (WH1) generates cytosolic amyloid particles that are inheritable within a bacterial lineage. Such amyloids trigger in bacteria a lethal cascade reminiscent to mitochondria impairment in human cells affected by neurodegeneration. To fulfil all the features of a prion-like protein, horizontal (intercellular) transmissibility remains to be demonstrated for RepA-WH1. Since this is experimentally intractable in bacteria, here we transiently expressed in a murine neuroblastoma cell line the soluble, barely cytotoxic RepA-WH1(WT) and assayed its response to co-incubation with in vitro assembled RepA-WH1(A31V) amyloid fibres. In parallel, cells releasing RepA-WH1(A31V) aggregates were co-cultured with human neuroblastoma cells expressing RepA-WH1(WT). Both the assembled fibres and the extracellular RepA-WH1(A31V) aggregates induce, in the cytosol of recipient cells, the formation of cytotoxic amyloid particles. Mass spectrometry analyses of the proteomes of both types of injured cells point to alterations in mitochondria, protein quality triage, signalling and intracellular traffic.\n\nSummary blurbThe horizontal, cell-to-cell spread of a bacterial prion-like protein is shown for the first time in mammalian cells. Amyloid cross-aggregation of distinct variants, and their associated toxicities, follow the same trend found in bacteria, underlining the universality of prion biology.

NADPH oxidases (NOXs) play a major role in the physiology of eukaryotic cells by mediating the production of reactive oxygen species (ROS). Evolutionarily distant proteins sharing the NOX catalytic core have been recently described in Bacteria. Among them, the Streptococcus pneumoniae NOX (SpNOX) has been proposed as a model for the study of NOXs due to its high activity and stability in detergent micelles. Here, we report high-resolution cryo-EM structures of substrate-free and stably reduced NADH-bound SpNOX, and of the NADPH-bound SpNOX and a Phe397Ala mutant under turnover conditions. In combination with structure-guided mutagenesis and biochemical analyses, we provide the structural basis for constitutive activity, the lack of substrate specificity towards NADPH and the electron transfer pathway. Additionally, we shed light on the catalytic regulation by the C-terminal tail residue Phe397 and the potential in vivo function of this protein.

Eukaryotes produce a large number of cytochrome P450s that mediate the synthesis and degradation of diverse endogenous and exogenous metabolites. Yet, most of these P450s are uncharacterized and global tools to study these challenging, membrane-resident enzymes remain to be exploited. Here, we applied activity profiling of plant, mouse and fungal P450s with chemical probes that become reactive when oxidized by P450 enzymes. Identification by mass spectrometry revealed labeling of a wide range of active P450s, including six plant P450s, 40 mouse P450s and 13 P450s of the fungal wheat pathogen Zymoseptoria tritici. We next used transient expression of GFP-tagged P450s by agroinfiltration to show ER-targeting and NADPH-dependent, activity-based labeling of plant, mouse and fungal P450s. Both global profiling and transient expression can be used to detect a broad range of active P450s to study e.g. their regulation and discover selective inhibitors.

Bacteria resist toxic arsenite (AsIII) in their environments by actively pumping the metalloid out of the cell via efflux pumps such as ArsB. However, the mechanism of extrusion remains poorly understood, which hinders the development of engineered bioremediation strategies. We report high-resolution cryo-EM structures of ArsB from the arsenic-tolerant bacterium Leptospirillum ferriphilum. ArsB adopts an inverted two-fold repeat architecture resembling that of other ion transporter (IT) superfamily proteins. Structures determined in the presence of AsIII and antimonite (SbIII) reveal that the metalloid substrates interact with polar residues at the core of the transmembrane domain primarily via hydrogen bonding. Mutagenesis and in vivo functional assays support these interactions. Our ArsB structures represent an inward-facing conformation, where the metalloid-binding site is exposed to the cytoplasm, suitable for metalloid capture. Furthermore, we demonstrate that AsIII resistance conferred by ArsB varies with external pH, supporting that ArsB is a proton (H+)-coupled secondary transporter. Mutagenesis, in vivo functional assays, and pKa estimation imply that conserved aspartate residues near the metalloid-binding site likely mediate the H+-coupling mechanism. Our findings provide structural insights into metalloid recognition and H+/metalloid antiport in ArsB, laying a foundation for further elucidation of the molecular basis of toxic metalloid detoxification in bacteria.

We report the cryoEM structure of the Francisella protein FTN_1118, identifying it as a novel 13 kDa periplasmic protein unique to the Francisella genus, which we now designate FPM13 (Francisella Periplasmic Metalloprotein, 13 kDa) based on its structural and biochemical properties. FPM13 was serendipitously identified during purification of Francisella type VI secretion system (T6SS) effector proteins, co-purifying with them. Its identity, initially unknown, was established using the novel cryoID method. The structure reveals a symmetrical, donut-shaped 18-mer with 9-fold dihedral symmetry, formed by two stacked nonamers head-to-head. It measures [~]8 nm both in height and in outer diameter, and has a 3.5 nm central channel. The complex features a double-layered wall with an inner {beta}-sheet core and an outer -helical shell. Each monomer adopts a compact fold comprising an N-terminus {beta}-strand, an -helix and two additional {beta} strands at the C-terminus. The assembly is stabilized by inter-ring loop interactions and hydrophobic and electrostatic contacts between neighboring subunits. Biochemical analyses, as shown by APEX-biotinylation and Triton X-114 phase partitioning, confirmed that FPM13 is a soluble periplasmic protein. ICP-MS demonstrated that FPM13 binds iron and copper. Deletion of FPM13 in Francisella novicida strains caused no growth defects in macrophages or mice but show increased copper sensitivity under iron-depleted conditions, suggesting a role for FPM13 in metal transport or detoxification.

The initiator (Inr) is the starting point for the transcription of many genes. Here, we generated highly predictive machine learning models of the human Inr region, and determined that the Inr is present in about 60% of natural promoters, identified a novel TATA-specific Inr, and detected the overlapping but functionally distinct TCT motif. Quantitative genome-wide analyses revealed a strict and synergistic interaction between the Inr and DPR, a duality between the TATA and DPR, a flexible and sometimes independent function of the TATA box in relation to the Inr, and different properties of the TCT motif in humans and Drosophila.

Lignocellulosic biomass is the most abundant and sustainable carbon source for bioproduction, but its efficient utilization is hampered by the heterogeneous mixture of sugars released upon hydrolysis. Most industrial strains consume these mixed sugars sequentially due to strong regulation and cross-inhibition, leading to complex processes and reduced carbon efficiency. To address this, we leverage a novel central metabolism that decouples central carbon flux from native regulatory feedback by employing a Gluconate-Bypass of glycolysis. We demonstrate that the Gluconate-Bypass effectively alleviates feedback regulation in E. coli, enabling co-consumption of glucose and xylose. Further strain engineering leads to the first robust co-utilization of four major lignocellulosic sugars: glucose, xylose, arabinose, and galactose. By decoupling central carbon flux from native regulatory feedback, this architecture provides a feedstock-agnostic platform that maintains high and robust consumption regardless of extreme fluctuations in sugar composition.

ALECT2 amyloidosis is a rare systemic disease characterized by the pathological deposition of leukocyte cell-derived chemotaxin-2 (LECT2) as amyloid fibrils, primarily affecting the kidneys and liver. The molecular mechanisms underlying LECT2 aggregation remain poorly defined, hindering diagnostic and therapeutic development. Here, we present cryo-electron microscopy structures of ex-vivo ALECT2 fibrils extracted from a patients kidney. We identified three fibril polymorphs: a predominant single-protofilament morphology and two minor double-protofilament morphologies. The dominant single-protofilament morphology comprises the full-length 133-residue LECT2 protein and retains all three native disulfide bonds. Low-resolution reconstructions of double-protofilament morphologies suggest they adopt a similar fold to the single protofilament morphology, but form paired assemblies with different inter-filament interfaces. Mass spectrometry also reveals acetylation within the fibrils. These findings offer critical insights into the structural basis of ALECT2 amyloid formation and identify molecular features that could inform future diagnostic and therapeutic approaches.

Bacterial biofilms underpin chronic infection and antimicrobial resistance, notably in Pseudomonas aeruginosa. Here we deconvolute a commercial alginate lyase preparation from Flavobacterium quisquiliarum and identify a previously uncharacterised ~21 kDa porphyrin-binding protein (FqPBP). Structural, biophysical and docking analyses reveal high-affinity tetrapyrrole binding. Recombinant FqPBP independently inhibits and disperses P. aeruginosa biofilms, implicating porphyrin sequestration and iron homeostasis in biofilm control and highlighting a potential therapeutic strategy targeting iron acquisition pathways.

Sporosarcina pasteurii is the most widely studied bacterium for microbially-induced calcium carbonate precipitation (MICP), a process of intense interest for materials and construction applications. Despite two decades of investigation, S. pasteurii has remained genetically intractable, limiting our mechanistic understanding of biomineralization pathways and constraining efforts to engineer scalable solutions. Here, we present the first genetic toolkit for S. pasteurii, including a stable replicating plasmid, a conjugation-based DNA delivery protocol, engineered inducible promoters, and methods for genome modification. Using homologous recombination, we precisely deleted 5.7 kb of the genome spanning two operons encoding urease activity and demonstrated complete loss of biocementation. We also screened a library of engineered transposon constructs for activity in S. pasteurii and generated a genome-wide mutant library with >15,000 unique insertion sites. Using this library, we identified putative genes affecting ureolytic growth, revealing previously inaccessible aspects of S. pasteurii genetics. This work establishes S. pasteurii as a genetically tractable platform for rational engineering of MICP and constitutes the first genetic modification capability within the Sporosarcina genus.