Biology subjects

Heddle, J. G.

Publications and source records attributed to Heddle, J. G..

8 recordsLinked to original sources

bioRxiv2026

Computational design of de novo integrated domains enables rational control of pathogen effector recognition in plant NLR immune receptors.

The rapid evolution of plant pathogens poses a persistent threat to global agricultural sustainability, often outpacing the discovery and deployment of natural disease resistance genes. While bioengineering of plant intracellular immune receptors (NLRs) offers a potential solution, developing bespoke immune recognition remains constrained by the laborious characterisation of natural receptors and plant-pathogen interactions. Here, we describe a programmable framework that leverages generative AI protein design tools, RFdiffusion and ProteinMPNN, to design de novo integrated domains (IDs) against diverse pathogen effectors. By integrating these bespoke binders into the modular rice blast Pik-1/Pik-2 NLR receptor chassis, we successfully engineer recognition of a non-cognate virulence factor (effector) from the Panama disease pathogen, Fusarium oxysporum f. sp. cubense Tropical Race 4. Functional assays in Nicotiana benthamiana demonstrate that these de novo domains facilitate specific effector perception and initiate immune signalling, while structural and biophysical analyses confirm that de novo integrated domains maintain high structural fidelity to the initial designs and associate with their targets via the predicted interaction interfaces. Additionally, our findings provide orthogonal evidence for the role of integrated domains in regulation of NLR signalling, demonstrating integration of de novo IDs can either trigger autoactivity or, in some cases, lead to effector-mediated repression of cell death. By decoupling immune perception from natural evolutionary history through deploying AI-designed sensory domains, this work establishes a design-lead framework for generation of programmable plant immune receptors, providing a new avenue for bioengineering crops against emerging pathogens.

plant biology↗

bioRxiv2025

High-Resolution Structures of Tobacco Mosaic Virus Disks from Cryo-Electron Microscopy

Tobacco mosaic virus has been involved in many important developments in virology, structural biology, and biotechnology. Despite decades of study, several key aspects of the viral assembly mechanism remain unclear, particularly the structure of the coat protein disk that initiates virus assembly by interacting with the viral RNA. Here we report the first cryo-electron microscopy structures of the coat protein disk under conditions typically used for in vitro virus assembly. We identify 1-, 2-, and 3-layered disks, all of which differ significantly from previous structures obtained by X-ray crystallography. These new models lead to a revised viral assembly mechanism. We also compare coat proteins produced in bacteria and plants to better understand the effect of N-terminal acetylation.

biochemistry↗

bioRxiv2024

Extended Plasmonic Nanostructures Templated by Tobacco Mosaic Virus Coat Protein

Optical and magnetic metamaterials possess interesting properties that cannot be achieved with conventional materials. However, there is currently no synthetic method offering both scalability and nanometer spatial precision. Biotemplating is a promising technique that has the potential to organize nanoscale components with high precision while being scalable and low-cost. Here we demonstrate a versatile template using hexahistidine-tagged tobacco mosaic virus coat protein. The protein self-assembles into disks which further assemble into extended nanostructures under mild conditions. Large sheets with either hexagonal or square packing and core-shell nanorods were formed, and gold nanoparticles were attached to the disks within each nanostructure to form assemblies of nanoparticle rings.

biochemistry↗

bioRxiv2024

Dynamic assembly of pentamer-based protein nanotubes

The molecular mechanisms and the geometrical theory underlying the polymorphic behavior of protein cages provide a basis for designing ones with the desired morphology and assembly properties. We show here that a circularly permuted cage-forming enzyme can controllably assemble into a variety of hollow spherical and cylindrical structures composed entirely of pentamers. A dramatic cage-to-tube transformation is facilitated by an untethered -helix domain that prevents the 3-fold symmetry interaction and imparts a torsion between the building blocks. The unique double- and triple-stranded helical arrangements of subunits are mathematically optimal tiling patterns for this type of pentamer. These structural insights afford guidelines for the design of customized protein nanotubes for smart delivery and nanoreactor systems.

bioengineering↗

bioRxiv2024

Structure of Escherichia coli DNA gyrase with chirally wrapped DNA supports ratchet-and-pawl mechanism for an ATP-powered supercoiling motor

Gyrase is essential for replication and transcription in bacteria, and as such is an important target for antibiotics including fluoroquinolones. Gyrase is a molecular machine that channels the energy of ATP hydrolysis into negative supercoiling of DNA. The mechanism proposed more than 40 years ago involves stabilising and inverting a chiral DNA loop; however, the molecular basis for this is poorly understood, as the loop was never directly observed. We present high-resolution cryoEM structures of the Escherichia coli gyrase - 217 bp DNA holocomplex, and of the moxifloxacin-bound gyrase complex with cleaved 217 bp DNA. Each structure constrains an intact figure-of-eight positively supercoiled DNA loop, poised for strand passage. The loop is stabilised by a GyrA {beta}-pinwheel domain which how we here show has a structure of a flat disc, and functions akin to a mini-nucleosome. Our data implies that during catalysis the ATPase domains of the enzyme undergo a large movement to push the transported DNA segment through the break in DNA. By comparing the catalytic site between native drug-free and moxifloxacin-bound gyrase structures that both contain a single metal ion we demonstrate that the enzyme is observed in a native pre-catalytic state. Finally, we propose a ratchet and pawl mechanism for energy coupling in gyrase. These unexpected findings call for re-evaluation of existing data and offer a framework for further experiments designed to dissect the details of how gyrase molecular motor converts chemical energy into mechanical tension.

molecular biology↗

bioRxiv2023

Reengineering of an artificial protein cage for efficient packaging of active enzymes

Protein cages that readily encapsulate active enzymes of interest present useful nanotools for delivery and catalysis, wherein those with programmable disassembly characteristics serve as particularly attractive platforms. Here we establish a general guest packaging system based on an artificial protein cage, TRAP-cage, the disassembly of which can be induced by the addition of reducing agents. In this system, TRAP-cage with SpyCatcher moieties in the lumen was prepared using genetic modification of the protein building block and assembled into a cage structure with either monovalent gold ions or molecular crosslinkers. The resulting protein cage can efficiently capture guest proteins equipped with a SpyTag by simply mixing them in aqueous solution. This post-assembly loading system which circumvents the exposure of guests to thiol-reactive crosslinkers, enables packaging of enzymes possessing a catalytic cysteine or a metal cofactor while retaining their catalytic activity.

bioengineering↗

bioRxiv2022

Gene amplifications cause high-level resistance against albicidin in Gram-negative bacteria

Antibiotic resistance is a continuously increasing concern for public health care. Understanding resistance mechanisms and their emergence is crucial for the development of new antibiotics and their effective use. Here, we report the discovery of a gene amplification-based mechanism that imparts an up to 1000-fold increase in resistance levels against the antibiotic albicidin. We show that this mechanism protects Salmonella Typhimurium and Escherichia coli by increasing the copy number of the GyrI-like transcription regulator STM3175 (YgiV) which binds albicidin. X-ray crystallography and molecular docking studies reveal a conserved binding motif that can interact with aromatic building blocks of albicidin. Phylogenetic studies suggest that this resistance mechanism is ubiquitous in Gram-negative bacteria and our experiments confirm that STM3175 homologs can convey resistance in pathogens such as Vibrio vulnificus and Pseudomonas aeruginosa.

microbiology↗

bioRxiv2020

Pentapeptide repeat proteins QnrB1 and AlbG require ATP hydrolysis to rejuvenate poisoned gyrase complexes

DNA gyrase, a type II topoisomerase found predominantly in bacteria, is the target for a variety of "poisons", namely natural product toxins (e.g. albicidin. microcin B17) and clinically important synthetic molecules (e.g. fluoroquinolones). Resistance to both groups can be mediated by pentapeptide repeat proteins (PRPs). Despite long-term studies, the mechanism of action of these protective PRPs is not known. We compared activities of two such proteins, QnrB1 and AlbG in vitro. Each of them provided specific protection against its cognate toxin (fluoroquinolone or albicidin), which strictly required ATP hydrolysis by gyrase. Through a combination of fluorescence anisotropy, pull-downs and photocrosslinking we show that QnrB1 binds to the GyrB protein. We further probed the QnrB1 binding site using site-specific incorporation of a photoreactive amino acid and mapped strong and specific crosslinks to the N-terminal ATPase/transducer domain. We propose a model in which protective PRPs bind to the enzyme as T-segment DNA mimics to promote dissociation of the bound poison molecule.

biochemistry↗