Evolution of endosymbiosis-mediated nuclear calcium signaling in land plants.

The ability of fungi to establish mycorrhizal associations with plants and enhance the acquisition of mineral nutrients stands out as a key feature of terrestrial life. Evidence indicates that arbuscular mycorrhizal (AM) association is a trait present in the common ancestor of land plants,1,2,3,4 suggesting that AM symbiosis was an important adaptation for plants in terrestrial environments.5 The activation of nuclear calcium signaling in roots is essential for AM within flowering plants.6 Given that the earliest land plants lacked roots, whether nuclear calcium signals are required for AM in non-flowering plants is unknown. To address this question, we explored the functional conservation of symbiont-induced nuclear calcium signals between the liverwort Marchantia paleacea and the legume Medicago truncatula. In M. paleacea, AM fungi penetrate the rhizoids and form arbuscules in the thalli.7 Here, we demonstrate that AM germinating spore exudate (GSE) activates nuclear calcium signals in the rhizoids of M. paleacea and that this activation is dependent on the nuclear-localized ion channel DOES NOT MAKE INFECTIONS 1 (MpaDMI1). However, unlike flowering plants, MpaDMI1-mediated calcium signaling is only required for the thalli colonization but not for the AM penetration within rhizoids. We further demonstrate that the mechanism of regulation of DMI1 has diverged between M. paleacea and M. truncatula, including a key amino acid residue essential to sustain DMI1 in an inactive state. Our study reveals functional evolution of nuclear calcium signaling between liverworts and flowering plants and opens new avenues of research into the mechanism of endosymbiosis signaling.

Bimodular architecture of bacterial effector SAP05 that drives ubiquitin-independent targeted protein degradation.

In eukaryotes, targeted protein degradation (TPD) typically depends on a series of interactions among ubiquitin ligases that transfer ubiquitin molecules to substrates leading to degradation by the 26S proteasome. We previously identified that the bacterial effector protein SAP05 mediates ubiquitin-independent TPD. SAP05 forms a ternary complex via interactions with the von Willebrand Factor Type A (vWA) domain of the proteasomal ubiquitin receptor Rpn10 and the zinc-finger (ZnF) domains of the SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) and GATA BINDING FACTOR (GATA) transcription factors (TFs). This leads to direct TPD of the TFs by the 26S proteasome. Here, we report the crystal structures of the SAP05-Rpn10vWA complex at 2.17 Å resolution and of the SAP05-SPL5ZnF complex at 2.20 Å resolution. Structural analyses revealed that SAP05 displays a remarkable bimodular architecture with two distinct nonoverlapping surfaces, a "loop surface" with three protruding loops that form electrostatic interactions with ZnF, and a "sheet surface" featuring two ß-sheets, loops, and α-helices that establish polar interactions with vWA. SAP05 binding to ZnF TFs involves single amino acids responsible for multiple contacts, while SAP05 binding to vWA is more stable due to the necessity of multiple mutations to break the interaction. In addition, positioning of the SAP05 complex on the 26S proteasome points to a mechanism of protein degradation. Collectively, our findings demonstrate how a small bacterial bimodular protein can bypass the canonical ubiquitin-proteasome proteolysis pathway, enabling ubiquitin-independent TPD in eukaryotic cells. This knowledge holds significant potential for the creation of TPD technologies.

Exploring the 5-Substituted 2-Aminobenzothiazole-Based DNA Gyrase B Inhibitors Active against ESKAPE Pathogens.

We present a new series of 2-aminobenzothiazole-based DNA gyrase B inhibitors with promising activity against ESKAPE bacterial pathogens. Based on the binding information extracted from the cocrystal structure of DNA gyrase B inhibitor A, in complex with Escherichia coli GyrB24, we expanded the chemical space of the benzothiazole-based series to the C5 position of the benzothiazole ring. In particular, compound E showed low nanomolar inhibition of DNA gyrase (IC50 < 10 nM) and broad-spectrum antibacterial activity against pathogens belonging to the ESKAPE group, with the minimum inhibitory concentration < 0.03 µg/mL for most Gram-positive strains and 4-16 µg/mL against Gram-negative E. coli, Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae. To understand the binding mode of the synthesized inhibitors, a combination of docking calculations, molecular dynamics (MD) simulations, and MD-derived structure-based pharmacophore modeling was performed. The computational analysis has revealed that the substitution at position C5 can be used to modify the physicochemical properties and antibacterial spectrum and enhance the inhibitory potency of the compounds. Additionally, a discussion of challenges associated with the synthesis of 5-substituted 2-aminobenzothiazoles is presented.

The CCP4 suite: integrative software for macromolecular crystallography.

The Collaborative Computational Project No. 4 (CCP4) is a UK-led international collective with a mission to develop, test, distribute and promote software for macromolecular crystallography. The CCP4 suite is a multiplatform collection of programs brought together by familiar execution routines, a set of common libraries and graphical interfaces. The CCP4 suite has experienced several considerable changes since its last reference article, involving new infrastructure, original programs and graphical interfaces. This article, which is intended as a general literature citation for the use of the CCP4 software suite in structure determination, will guide the reader through such transformations, offering a general overview of the new features and outlining future developments. As such, it aims to highlight the individual programs that comprise the suite and to provide the latest references to them for perusal by crystallographers around the world.

Resurrection of plant disease resistance proteins via helper NLR bioengineering.

Parasites counteract host immunity by suppressing helper nucleotide binding and leucine-rich repeat (NLR) proteins that function as central nodes in immune receptor networks. Understanding the mechanisms of immunosuppression can lead to strategies for bioengineering disease resistance. Here, we show that a cyst nematode virulence effector binds and inhibits oligomerization of the helper NLR protein NRC2 by physically preventing intramolecular rearrangements required for activation. An amino acid polymorphism at the binding interface between NRC2 and the inhibitor is sufficient for this helper NLR to evade immune suppression, thereby restoring the activity of multiple disease resistance genes. This points to a potential strategy for resurrecting disease resistance in crop genomes.

New Dual Inhibitors of Bacterial Topoisomerases with Broad-Spectrum Antibacterial Activity and In Vivo Efficacy against Vancomycin-Intermediate Staphylococcus aureus.

A new series of dual low nanomolar benzothiazole inhibitors of bacterial DNA gyrase and topoisomerase IV were developed. The resulting compounds show excellent broad-spectrum antibacterial activities against Gram-positive Enterococcus faecalis, Enterococcus faecium and multidrug resistant (MDR) Staphylococcus aureus strains [best compound minimal inhibitory concentrations (MICs): range, <0.03125-0.25 µg/mL] and against the Gram-negatives Acinetobacter baumannii and Klebsiella pneumoniae (best compound MICs: range, 1-4 µg/mL). Lead compound 7a was identified with favorable solubility and plasma protein binding, good metabolic stability, selectivity for bacterial topoisomerases, and no toxicity issues. The crystal structure of 7a in complex with Pseudomonas aeruginosa GyrB24 revealed its binding mode at the ATP-binding site. Expanded profiling of 7a and 7h showed potent antibacterial activity against over 100 MDR and non-MDR strains of A. baumannii and several other Gram-positive and Gram-negative strains. Ultimately, in vivo efficacy of 7a in a mouse model of vancomycin-intermediate S. aureus thigh infection was also demonstrated.

The CTP-binding domain is disengaged from the DNA-binding domain in a cocrystal structure of Bacillus subtilis Noc-DNA complex.

In Bacillus subtilis, a ParB-like nucleoid occlusion protein (Noc) binds specifically to Noc-binding sites (NBSs) on the chromosome to help coordinate chromosome segregation and cell division. Noc does so by binding to CTP to form large membrane-associated nucleoprotein complexes to physically inhibit the assembly of the cell division machinery. The site-specific binding of Noc to NBS DNA is a prerequisite for CTP-binding and the subsequent formation of a membrane-active DNA-entrapped protein complex. Here, we solve the structure of a C-terminally truncated B. subtilis Noc bound to NBS DNA to reveal the conformation of Noc at this crucial step. Our structure reveals the disengagement between the N-terminal CTP-binding domain and the NBS-binding domain of each DNA-bound Noc subunit; this is driven, in part, by the swapping of helices 4 and 5 at the interface of the two domains. Site-specific crosslinking data suggest that this conformation of Noc-NBS exists in solution. Overall, our results lend support to the recent proposal that parS/NBS binding catalyzes CTP binding and DNA entrapment by preventing the reengagement of the CTP-binding domain and the DNA-binding domain from the same ParB/Noc subunit.

Discovery and Hit-to-Lead Optimization of Benzothiazole Scaffold-Based DNA Gyrase Inhibitors with Potent Activity against Acinetobacter baumannii and Pseudomonas aeruginosa.

We have developed compounds with a promising activity against Acinetobacter baumannii and Pseudomonas aeruginosa, which are both on the WHO priority list of antibiotic-resistant bacteria. Starting from DNA gyrase inhibitor 1, we identified compound 27, featuring a 10-fold improved aqueous solubility, a 10-fold improved inhibition of topoisomerase IV from A. baumannii and P. aeruginosa, a 10-fold decreased inhibition of human topoisomerase IIα, and no cross-resistance to novobiocin. Cocrystal structures of 1 in complex with Escherichia coli GyrB24 and (S)-27 in complex with A. baumannii GyrB23 and P. aeruginosa GyrB24 revealed their binding to the ATP-binding pocket of the GyrB subunit. In further optimization steps, solubility, plasma free fraction, and other ADME properties of 27 were improved by fine-tuning of lipophilicity. In particular, analogs of 27 with retained anti-Gram-negative activity and improved plasma free fraction were identified. The series was found to be nongenotoxic, nonmutagenic, devoid of mitochondrial toxicity, and possessed no ion channel liabilities.

A novel mutant allele of AtCNGC15 reveals a dual function of nuclear calcium release in the root meristem.

Calcium release to the nucleoplasm of root meristem cells was demonstrated to modulate root development. The calcium channel encoded by cyclic nucleotide-gated channel (CNGC) 15 localizes at the nuclear envelope in young Arabidopsis seedlings. In contrast, at later stages of root growth, overexpression analysis showed that AtCNGC15 can relocalize to the plasma membrane to mediate primary nitrate-induced gene expression. This raises the question as to whether nuclear localized AtCNGC15 is required for root apical meristem development in young Arabidopsis seedlings, and whether nitrate signalling occurs independently of nuclear localized AtCNGC15 at this developmental stage. In this study, we characterize a novel mutant allele of AtCNGC15 and demonstrate that the mutation of a highly conserved aspartic acid in the C-linker domain is sufficient to impair the gating of AtCNCG15. We demonstrate that AtCNGC15 mediates the nuclear calcium release that modulates root apical meristem development and nitrate-induced LBD39 expression. We also show that, in the presence of nitrate, the relocalization of AtCNGC15 at the plasma membrane occurs specifically in the columella cells. Our results further suggest that the induction of LBD37, LBD38, and LBD39 in the presence of nitrate is modulated by different inputs of cytoplasmic or nuclear calcium release.

Structural insights into the mechanism of adaptive ribosomal modification by Pseudomonas RimK.

Bacteria are equipped with a diverse set of regulatory tools that allow them to quickly adapt to their environment. The RimK system allows for Pseudomonas spp. to adapt through post-transcriptional regulation by altering the ribosomal subunit RpsF. RimK is found in a wide range of bacteria with a conserved amino acid sequence, however, the genetic context and the role of this protein is highly diverse. By solving and comparing the structures of RimK homologs from two related but functionally divergent systems, we uncovered key structural differences that likely contribute to the different activity levels of each of these homologs. Moreover, we were able to clearly resolve the active site of this protein for the first time, resolving binding of the glutamate substrate. This work advances our understanding of how subtle differences in protein sequence and structure can have profound effects on protein activity, which can in turn result in widespread mechanistic changes.

Expansion of the Catalytic Repertoire of Alcohol Dehydrogenases in Plant Metabolism.

Medium-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,ß-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.

A blast fungus zinc-finger fold effector binds to a hydrophobic pocket in host Exo70 proteins to modulate immune recognition in rice.

Exocytosis plays an important role in plant-microbe interactions, in both pathogenesis and symbiosis. Exo70 proteins are integral components of the exocyst, an octameric complex that mediates tethering of vesicles to membranes in eukaryotes. Although plant Exo70s are known to be targeted by pathogen effectors, the underpinning molecular mechanisms and the impact of this interaction on infection are poorly understood. Here, we show the molecular basis of the association between the effector AVR-Pii of the blast fungus Maganaporthe oryzae and rice Exo70 alleles OsExo70F2 and OsExo70F3, which is sensed by the immune receptor pair Pii via an integrated RIN4/NOI domain. The crystal structure of AVR-Pii in complex with OsExo70F2 reveals that the effector binds to a conserved hydrophobic pocket in Exo70, defining an effector/target binding interface. Structure-guided and random mutagenesis validates the importance of AVR-Pii residues at the Exo70 binding interface to sustain protein association and disease resistance in rice when challenged with fungal strains expressing effector mutants. Furthermore, the structure of AVR-Pii defines a zinc-finger effector fold (ZiF) distinct from the MAX (Magnaporthe Avrs and ToxB-like) fold previously described for a majority of characterized M. oryzae effectors. Our data suggest that blast fungus ZiF effectors bind a conserved Exo70 interface to manipulate plant exocytosis and that these effectors are also baited by plant immune receptors, pointing to new opportunities for engineering disease resistance.

An allelic series of starch-branching enzyme mutants in pea (Pisum sativum L.) reveals complex relationships with seed starch phenotypes.

A set of mutant pea lines carrying induced mutations within the major seed-expressed starch-branching enzyme gene has been characterised at the molecular, chemical and agronomic levels. Eight of the induced mutations, three of which predicted a premature stop codon, were compared with the naturally occurring starch-branching enzyme mutation within the same genetic background. Starch, amylose and sugar measurements, coupled with analysis by ultra-high performance liquid chromatography-size exclusion chromatography of starches, identified a range of phenotypes which were grouped according to the nature of the mutation. Homology modelling of proteins supported the differences in phenotypes observed. Differences in field performance were evident for selected mutants, particularly in seed yield and mean seed weight traits for early compared with late spring sowings. The data show the potential of an allelic series of mutants at this locus for nutritional studies. CHEMICAL COMPOUNDS: starch, amylose, amylopectin, raffinose, stachyose, verbascose.

A CTP-dependent gating mechanism enables ParB spreading on DNA.

Proper chromosome segregation is essential in all living organisms. The ParA-ParB-parS system is widely employed for chromosome segregation in bacteria. Previously, we showed that Caulobacter crescentus ParB requires cytidine triphosphate to escape the nucleation site parS and spread by sliding to the neighboring DNA (Jalal et al., 2020). Here, we provide the structural basis for this transition from nucleation to spreading by solving co-crystal structures of a C-terminal domain truncated C. crescentus ParB with parS and with a CTP analog. Nucleating ParB is an open clamp, in which parS is captured at the DNA-binding domain (the DNA-gate). Upon binding CTP, the N-terminal domain (NTD) self-dimerizes to close the NTD-gate of the clamp. The DNA-gate also closes, thus driving parS into a compartment between the DNA-gate and the C-terminal domain. CTP hydrolysis and/or the release of hydrolytic products are likely associated with reopening of the gates to release DNA and recycle ParB. Overall, we suggest a CTP-operated gating mechanism that regulates ParB nucleation, spreading, and recycling.

CTP regulates membrane-binding activity of the nucleoid occlusion protein Noc.

ATP- and GTP-dependent molecular switches are extensively used to control functions of proteins in a wide range of biological processes. However, CTP switches are rarely reported. Here, we report that a nucleoid occlusion protein Noc is a CTPase enzyme whose membrane-binding activity is directly regulated by a CTP switch. In Bacillus subtilis, Noc nucleates on 16 bp NBS sites before associating with neighboring non-specific DNA to form large membrane-associated nucleoprotein complexes to physically occlude assembly of the cell division machinery. By in vitro reconstitution, we show that (1) CTP is required for Noc to form the NBS-dependent nucleoprotein complex, and (2) CTP binding, but not hydrolysis, switches Noc to a membrane-active state. Overall, we suggest that CTP couples membrane-binding activity of Noc to nucleoprotein complex formation to ensure productive recruitment of DNA to the bacterial cell membrane for nucleoid occlusion activity.

Two NLR immune receptors acquired high-affinity binding to a fungal effector through convergent evolution of their integrated domain.

A subset of plant NLR immune receptors carry unconventional integrated domains in addition to their canonical domain architecture. One example is rice Pik-1 that comprises an integrated heavy metal-associated (HMA) domain. Here, we reconstructed the evolutionary history of Pik-1 and its NLR partner, Pik-2, and tested hypotheses about adaptive evolution of the HMA domain. Phylogenetic analyses revealed that the HMA domain integrated into Pik-1 before Oryzinae speciation over 15 million years ago and has been under diversifying selection. Ancestral sequence reconstruction coupled with functional studies showed that two Pik-1 allelic variants independently evolved from a weakly binding ancestral state to high-affinity binding of the blast fungus effector AVR-PikD. We conclude that for most of its evolutionary history the Pik-1 HMA domain did not sense AVR-PikD, and that different Pik-1 receptors have recently evolved through distinct biochemical paths to produce similar phenotypic outcomes. These findings highlight the dynamic nature of the evolutionary mechanisms underpinning NLR adaptation to plant pathogens.

Plant-expressed virus-like particles reveal the intricate maturation process of a eukaryotic virus.

Many virus capsids undergo exquisitely choreographed maturation processes in their host cells to produce infectious virions, and these remain poorly understood. As a tool for studying virus maturation, we transiently expressed the capsid protein of the insect virus Nudaurelia capensis omega virus (NωV) in Nicotiana benthamiana and were able to purify both immature procapsids and mature capsids from infiltrated leaves by varying the expression time. Cryo-EM analysis of the plant-produced procapsids and mature capsids to 6.6 Å and 2.7 Å resolution, respectively, reveals that in addition to large scale rigid body motions, internal regions of the subunits are extensively remodelled during maturation, creating the active site required for autocatalytic cleavage and infectivity. The mature particles are biologically active in terms of their ability to lyse membranes and have a structure that is essentially identical to authentic virus. The ability to faithfully recapitulate and visualize a complex maturation process in plants, including the autocatalytic cleavage of the capsid protein, has revealed a ~30 Å translation-rotation of the subunits during maturation as well as conformational rearrangements in the N and C-terminal helical regions of each subunit.

Analysis of Protein-DNA Interactions Using Surface Plasmon Resonance and a ReDCaT Chip.

The recognition of specific DNA sequences by proteins is crucial to fundamental biological processes such as DNA replication, transcription, and gene regulation. The technique of surface plasmon resonance (SPR) is ideally suited for the measurement of these interactions because it is quantitative, simple to implement, reproducible, can be automated, and requires very little sample. This typically involves the direct capture of biotinylated DNA to a streptavidin (SA) chip before flowing over the protein of interest and monitoring the interaction. However, once the DNA has been immobilized on the chip, it cannot be removed without damaging the chip surface. Moreover, if the protein-DNA interaction is strong, then it may not be possible to remove the protein from the DNA without damaging the chip surface. Given that the chips are costly, this will limit the number of samples that can be tested. Therefore, we have developed a Reusable DNA Capture Technology, or ReDCaT chip, that enables a single streptavidin chip to be used multiple times making the technique simple, quick, and cost effective. The general steps to prepare the ReDCaT chip, run a simple binding experiment, and analysis of data will be described in detail. Some additional applications will also be introduced.

The pentapeptide-repeat protein, MfpA, interacts with mycobacterial DNA gyrase as a DNA T-segment mimic.

DNA gyrase, a type II topoisomerase, introduces negative supercoils into DNA using ATP hydrolysis. The highly effective gyrase-targeted drugs, fluoroquinolones (FQs), interrupt gyrase by stabilizing a DNA-cleavage complex, a transient intermediate in the supercoiling cycle, leading to double-stranded DNA breaks. MfpA, a pentapeptide-repeat protein in mycobacteria, protects gyrase from FQs, but its molecular mechanism remains unknown. Here, we show that Mycobacterium smegmatis MfpA (MsMfpA) inhibits negative supercoiling by M. smegmatis gyrase (Msgyrase) in the absence of FQs, while in their presence, MsMfpA decreases FQ-induced DNA cleavage, protecting the enzyme from these drugs. MsMfpA stimulates the ATPase activity of Msgyrase by directly interacting with the ATPase domain (MsGyrB47), which was confirmed through X-ray crystallography of the MsMfpA-MsGyrB47 complex, and mutational analysis, demonstrating that MsMfpA mimics a T (transported) DNA segment. These data reveal the molecular mechanism whereby MfpA modulates the activity of gyrase and may provide a general molecular basis for the action of other pentapeptide-repeat proteins.

Potent DNA gyrase inhibitors bind asymmetrically to their target using symmetrical bifurcated halogen bonds.

Novel bacterial type II topoisomerase inhibitors (NBTIs) stabilize single-strand DNA cleavage breaks by DNA gyrase but their exact mechanism of action has remained hypothetical until now. We have designed a small library of NBTIs with an improved DNA gyrase-binding moiety resulting in low nanomolar inhibition and very potent antibacterial activity. They stabilize single-stranded cleavage complexes and, importantly, we have obtained the crystal structure where an NBTI binds gyrase-DNA in a single conformation lacking apparent static disorder. This directly proves the previously postulated NBTI mechanism of action and shows that they stabilize single-strand cleavage through asymmetric intercalation with a shift of the scissile phosphate. This crystal stucture shows that the chlorine forms a halogen bond with the backbone carbonyls of the two symmetry-related Ala68 residues. To the best of our knowledge, such a so-called symmetrical bifurcated halogen bond has not been identified in a biological system until now.