Biology subjects

Lawson, D. M.

Publications and source records attributed to Lawson, D. M..

14 recordsLinked to original sources

bioRxiv2025

An effector from the potato late blight pathogen bridges ENTH-domain protein TOL9a to an activated helper NLR to suppress immunity

Pathogens counteract central nodes of NLR immune receptor networks to suppress immunity. However, the mechanisms by which pathogens hijack helper NLR pathways are poorly understood. Here, we show that an effector from the potato late blight pathogen Phytophthora infestans bridges the host protein NbTOL9a, a putative member of the host ESCRT pathway, to a helper NLR to suppress immunity. In this work, we solved the crystal structure of the RXLR-LWY effector AVRcap1b in complex with the ENTH domain of NbTOL9a. The structure revealed that unlike other RXLR-LWY effectors, AVRcap1b has a novel L-shaped fold that defines a new structural family of effectors in the Phytophthora genus. Moreover, we defined the AVRcap1b/NbTOL9a binding interface and designed effector mutants that dont bind NbTOL9a, impairing immune suppression. This indicates that ENTH binding is required for full virulence activity of this effector. Lastly, we show that AVRcap1b associates specifically with activated NbNRC2 independently of NbTOL9a binding. This suggests that the effector functions as a bridge that interconnects NbNRC2 with the NbTOL9a pathway. These results illustrate an unprecedented pathogen mechanism to hijack helper NLR pathways and suppress immunity.

plant biology↗

bioRxiv2025

Structural analysis of the NifL-NifA complex reveals the molecular basis of anti-activation of nitrogen fixation gene expression in Azotobacter vinelandii

Understanding the molecular basis of regulated nitrogen (N2) fixation is essential for engineering N2-fixing bacteria that fulfill the demand of crop plants for fixed nitrogen, reducing our reliance on synthetic nitrogen fertilizers. In Azotobacter vinelandii and many other members of Proteobacteria, the two-component system NifL-NifA controls the expression of nif genes that encode the nitrogen fixation machinery. The NifL-NifA system evolved the ability to integrate several environmental cues, such as oxygen, nitrogen, and carbon availability. The nitrogen fixation machinery is thereby only activated under strictly favorable conditions, enabling diazotrophs to thrive in competitive environments. Whilst genetic and biochemical studies have enlightened our understanding of how NifL represses NifA, the molecular basis of NifA sequestration by NifL depends on structural information on their interaction. Here, we present mechanistic insights into how nitrogen fixation is regulated by combining biochemical and genetic approaches with a low-resolution cryo-EM map of the oxidized NifL-NifA complex. Our findings define the interaction surface between NifL and NifA and reveal how this interaction can be manipulated to generate bacterial strains with increased nitrogen fixation rates able to secrete surplus nitrogen outside the cell, a crucial step in engineering improved nitrogen delivery to crop plants.

microbiology↗

bioRxiv2024

Unravelling the Maturation Pathway of a Eukaryotic Virus through Cryo-EM

The importance of virus maturation has been appreciated for nearly 70 years 1 as it provides models for large-scale protein reorganization resulting in functional activation as well as being a target for antiviral therapies 2. However, a detailed description of the pathway from the initial assembly product (procapsid) to the mature, infectious particle (virion) has been elusive. This is due to the "in cell" nature of the natural process, the 2- state behavior of maturation (no detectable intermediates) in some viruses in vitro 3 and heterogeneous populations of particle intermediates that are only partially matured in other systems 4. The non-enveloped, T=4, ssRNA-containing, Nudaurelia capensis omega virus (N{omega}V), is a highly accessible model system that exemplifies the maturation process of a eukaryotic virus. During maturation the particle shrinks in outer diameter from 482 [A] (pH 7.5) to 428 [A] (pH 5.0). It is possible to mimic the maturation process in vitro by lowering the pH of a population of procapsids produced in heterologous systems5. Indeed, by controlling the pH in vitro it is possible to produce homogenous populations of intermediate N{omega}V virus-like particles (VLPs) that occur too fleetingly to be observed in vivo 6. Here we report structural models, based on cryo-electron microscopy (cryo-EM), of five intermediates in the N{omega}V maturation process. The structures of the intermediate particles reveal unique, quaternary position-dependent trajectories and refolding of subunit N and C-terminal regions, including the formation of the autocatalytic cleavage site at N570. The detailed structures reported here, coupled with previously determined structures of the procapsids and mature particles, allows the maturation pathway to be described in detail for the first time for a eukaryotic virus.

biochemistry↗

bioRxiv2024

Molecular switching of a DNA-sliding clamp to a repressor mediates long-range gene silencing

Long-range gene regulation is rare in bacteria and is confined to the classical DNA looping model. Here, we use a combination of biophysical approaches, including X-ray crystallography and single-molecule analysis, to show that long-range gene silencing on the plasmid RK2, a source of multidrug resistance across diverse Gram-negative bacteria, is achieved cooperatively by a DNA-sliding clamp, KorB, and a clamp-locking protein, KorA. We find that KorB is a CTPase clamp that can entrap and slide along DNA to reach distal target promoters. We resolved the tripartite crystal structure of a KorB-KorA-DNA co-complex, revealing that KorA latches KorB into a closed-clamp state. KorA thus stimulates repression by stalling KorB sliding at target promoters to occlude RNA polymerase holoenzymes. Altogether, our findings explain the mechanistic basis for KorB role-switching from a DNA-sliding clamp to a co-repressor, and provide a new paradigm for the long-range regulation of gene expression.

microbiology↗

bioRxiv2023

Activation of plant immunity through conversion of a helper NLR homodimer into a resistosome

Nucleotide-binding domain and leucine-rich repeat (NLR) proteins can engage in complex interactions to detect pathogens and execute a robust immune response via downstream helper NLRs. However, the biochemical mechanisms of helper NLR activation by upstream sensor NLRs remain poorly understood. Here, we show that the coiled-coil helper NLR NRC2 accumulates in vivo as a homodimer that converts into a higher order oligomer upon activation by its upstream virus disease resistance protein Rx. The Cryo-EM structure of NRC2 in its resting state revealed intermolecular interactions that mediate homodimer formation. These dimerization interfaces have diverged between paralogous NRC proteins to insulate critical network nodes and enable redundant immune pathways. Our results expand the molecular mechanisms of NLR activation pointing to transition from homodimers to higher-order oligomeric resistosomes.

plant biology↗

bioRxiv2023

Bimodular Architecture of Bacterial Effector SAP05 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 discovered 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-vWA complex at 2.17 [A] resolution and of the SAP05-ZnF(SPL5) complex at 2.20 [A] resolution. Structural analyses revealed that SAP05 displays a unique bimodular architecture with two distinct non-overlapping surfaces, a loop surface with three protruding loops that form electrostatic interactions with ZnF, and a sheet surface featuring two {beta}-sheets, loops and -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 UPS cellular proteolysis pathway, enabling ubiquitin-independent TPD in eukaryotic cells. This knowledge holds significant potential for the creation of novel TPD technologies.

biochemistry↗

bioRxiv2022

Resurrection of plant disease resistance proteins via helper NLR bioengineering

Parasites counteract host immunity by suppressing helper 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. A single 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 novel strategy for resurrecting disease resistance in crop genomes. One sentence summaryA helper NLR is mutated to evade inhibition by a parasite effector.

plant biology↗

bioRxiv2022

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 ,{beta}-unsaturated iminium moiety. Comparison with structures of plant-derived ADHs that catalyse 1,2-aldehyde and 1,2-iminium reductions suggest how the canonical ADH active site can be modified to carry out atypical carbonyl reductions, providing insight into how chemical reactions are diversified in plant metabolism.

biochemistry↗

bioRxiv2022

Binding of a blast fungus Zinc-finger fold effector to a hydrophobic pocket in the host exocyst subunit Exo70 modulates immune recognition in rice

Exocytosis plays an important role in plant-microbe interactions, both in 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 is 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 a new 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. Further, the structure of AVR-Pii defines a novel Zinc- finger effector fold (ZiF) distinct from the MAX fold previously described for the majority of characterized M. oryzae effectors. Our data suggests 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. Significance statementPlant diseases destroy [~]20-30% of annual crop production, contributing to global food insecurity. Discovering how pathogen effectors target host proteins to promote virulence is essential for understanding pathogenesis and can be used for developing disease resistant crops. Here, we reveal the structural basis of how an effector from the blast pathogen (AVR-Pii) binds a specific host target (rice Exo70), and how this underpins immune recognition. This has implications for understanding the molecular mechanisms of blast disease and for the engineering of new recognition specificities in plant immune receptors to confer resistance to a major crop pathogen.

plant biology↗

bioRxiv2022

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

In Bacillus subtilis, a ParB-like nucleoid occlusion protein (Noc) binds specifically to Noc-binding sites (NBS) around the chromosome to help coordinate chromosome segregation and cell division. Noc does so by binding to cytidine triphosphate (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 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 re-engagement of the NTD and DBD from the same ParB/Noc subunit.

microbiology↗

bioRxiv2021

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 (i) CTP is required for Noc to form the NBS-dependent nucleoprotein complex, and (ii) 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.

microbiology↗

bioRxiv2021

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 biology↗

bioRxiv2019

Structural and biochemical analyses of Caulobacter crescentus ParB reveal the role of its N-terminal domain in chromosome segregation

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 1. 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 re-opening of the gates to release DNA and to recycle ParB. Overall, we suggest a CTP-operated gating mechanism that regulates ParB nucleation, spreading, and recycling.

microbiology↗

bioRxiv2019

Evolving a new protein-DNA interface via sequential introduction of permissive and specificity-switching mutations

Specific interactions between proteins and DNA are essential to many biological processes. Yet it remains unclear how the diversification in DNA-binding specificity was brought about, and what were the mutational paths that led to changes in specificity. Using a pair of evolutionarily related DNA-binding proteins, each with a different DNA preference (ParB and Noc: both having roles in bacterial chromosome maintenance), we show that specificity is encoded by a set of four residues at the protein-DNA interface. Combining X-ray crystallography and deep mutational scanning of the interface, we suggest that permissive mutations must be introduced before specificity-switching mutations to reprogram specificity, and that mutational paths to a new specificity do not necessarily involve dual-specificity intermediates. Overall, our results provide insight into the possible evolutionary history of ParB and Noc, and in a broader context, might be useful in understanding the evolution of other classes of DNA-binding proteins.

microbiology↗