Katherine Stott

Tau transitions from a functional microtubule-stabilising monomer into pathological aggregates in tauopathies. However, the molecular mechanism by which soluble Tau transitions into insoluble aggregates remains poorly understood. Here, we show that modulation of Tau’s internal charge-charge interactions by pH and ionic strength is sufficient to remodel its monomeric conformational ensemble and direct its subsequent self-assembly pathway. By subjecting the expanded random coil-like 2N4R Tau to prolonged incubation at low ionic strength and near its isoelectric point, we generated a long-lived compact molten globule-like monomer. Although both expanded and compacted conformations remain disordered, they differ markedly in their local solvent accessibility and dynamics. We show that monomer compaction is stabilised by weak interactions that remodel the solvent accessibility of the microtubule-binding repeat region and its flanking sequences. Consequently, these conformational changes alter Tau phase behaviour, inhibiting liquid-liquid phase separation and amyloid fibril formation. Our findings demonstrate that charge-driven remodelling of the Tau conformational ensemble determines its phase behaviour and provide a mechanistic insight into the molecular events underlying tauopathies.

Pseudomonas aeruginosa relies on heme acquisition to sustain growth and virulence in the iron-limited environment of the host, particularly in the acidic airways of patients with cystic fibrosis where antibiotic efficacy is markedly reduced. The Has heme assimilation system is initiated by the secreted hemophore HasA, which binds extracellular heme and delivers it to the outer-membrane receptor HasR. During infection, HasA is proteolytically processed, generating a truncated form that constitutes the biologically relevant species. Here, we characterise the structural and functional properties of truncated HasA under disease-associated acidic conditions (pH 6.5) and perform a rationally designed Systemic Evolution of Ligands by EXponential enrichment (SELEX) method to identify DNA aptamers capable of binding this hemophore. Biophysical analyses revealed that truncated HasA is folded, displays a mixed α/β secondary structure, and exists as a concentration-dependent mixture of monomers and domain-swapped dimers. Circular dichroism and nano-differential scanning fluorimetry identified two thermal transitions, consistent with the coexistence of apo/holo and monomer/dimer species. The apo protein bound heme with high affinity ( K _D = 113 nM) and a 1:1 stoichiometry, confirming preservation of its functional binding mechanism. Twelve rounds of SELEX, incorporating target switching and platform switching, yielded a highly enriched aptamer pool dominated by two sequences, HasA_1 and HasA_2. ELONA (Enzyme-Linked Oligonucleotide Assay) assays demonstrated that both aptamers bind specifically to truncated HasA, and biolayer interferometry revealed nanomolar dissociation constants ( K _D = 0.59 µM and 0.32 µM, respectively). These aptamers did not bind full-length HasA, BSA, or control sequences, confirming that selection under acidic conditions drove specificity toward the physiologically relevant form of the hemophore. Our findings identify HasA-binding DNA aptamers that retain function in acidic environments where antibiotic potency is compromised, highlighting their potential as molecular tools to disrupt heme acquisition in P. aeruginosa . This work establishes a foundation for developing aptamer-based antimicrobial strategies targeting the Has system, particularly under acidic conditions where conventional antibiotics are compromised.

TonB-dependent uptake systems of Gram-negative bacterial pathogens constitute prominent virulence factors, allowing nutrient acquisition, primarily siderophore-bound iron, to cross the highly impermeable outer membrane (OM). Remarkably, the ferredoxin uptake system (Fus) of Pectobacteriaceae , a group of soft rot-inducing plant pathogens, imports an entire folded host protein into the periplasm and extracts its bound iron for growth. The inner membrane protein FusB, a TonB homologue, plays two roles in facilitating ferredoxin import. First, like other TonBs, it remodels the globular plug domain obstructing the lumen of the OM receptor FusA to allow ferredoxin passage. Unusually for a TonB protein, FusB then interacts directly with the FusA-bound ferredoxin substrate to facilitate its transport into the periplasm. In this work, we describe structures of FusB-ferredoxin and homodimeric FusB complexes and determine the key features of the binding interfaces formed by FusB with FusA and ferredoxin. We postulate that under resting conditions FusB exists a homodimer, stabilised by an intermolecular R241-D322 salt bridge. The homodimer dissociates when the “FusB-box” of FusA outcompetes one protomer, and FusA D53 displaces FusB D322. Upon ferredoxin binding, FusB undergoes a structural rearrangement, expanding its β-sheet from three to four strands. In agreement with the proposed sequence of events, ferredoxin binding displaces the receptor (FusA) from FusB with Arg241 forming an intramolecular salt bridge with Asp322 to stabilise the newly formed β-hairpin of FusB. We propose a mechanistic model for ferredoxin import in which FusB Arg241 acts as a molecular switch, and two distinct regions function as interaction hotspots.

Summary: Plant primary and secondary cell walls differ in molecular composition, structure, and mechanical properties. While secondary wall xylan has been extensively characterised, the structure of xylan in primary walls remains less well understood, particularly in gymnosperms. Here, we identify a previously uncharacterised β-1,2-linked xylosyl side chain in conifer and Arabidopsis thaliana xylan. Using enzymatic fingerprinting, NMR, and mass spectrometry, we show that this structure is positioned two xylose residues away from glucuronic acid substitutions, forming an evenly patterned substituted xylan. This spacing pattern is consistent with xylan–cellulose interaction, suggesting a structural role in primary wall architecture. This modification, found in primary wall-rich tissues of diverse conifer species, including needles and pro-embryogenic mass (PEM), is also present in Arabidopsis callus. We demonstrate that conifer Group III GT61 glycosyltransferases introduce this modification with consistent positional specificity. In Arabidopsis, three closely related GT61 enzymes act redundantly to generate the same structure, and their combined loss results in its complete absence. These findings uncover a conserved primary wall xylan modification in seed plants and define the GT61 enzymes responsible for its biosynthesis, opening new avenues to explore how xylan structure contributes to primary wall function. Significance Statement: Xylan structure is well characterised in secondary walls, but its primary wall counterpart remains poorly understood. We identified a conserved β-1,2-xylosyl modification on xylan in the primary walls of conifers and Arabidopsis. This side chain is positioned at a defined position from a glucuronic acid substitution and is introduced by GT61 glycosyltransferases that cluster in one phylogenetic subclade. Our findings revealed a previously unrecognised xylan structural pattern and the biosynthetic enzymes responsible for its addition. This work expands the current understanding of primary wall architecture across seed plants.

The endoplasmic reticulum (ER) unfolded protein response (UPR) is tuned by the balance between unfolded proteins and chaperones. While reserve chaperones are known to suppress the UPR transducers via their stress-sensing luminal domains, the underlying structural mechanisms remain unclear. Cellular and biophysical analyses established that the ER chaperone AGR2 forms a repressive complex with the luminal domain of the UPR transducer IRE1β. Structural prediction, X-ray crystallography and NMR spectroscopy identify critical interactions between an AGR2 monomer and a regulatory loop in IRE1β’s luminal domain. However, in the repressive complex it is an AGR2 dimer that binds IRE1β. Cryo-EM reconstruction reveals a mechanism of unanticipated simplicity: one AGR2 protomer engages the regulatory loop, while the second asymmetrically binds IRE1β’s luminal domain’s C-terminus, blocking IRE1β-activating dimerization. Molecular dynamic simulations indicate that the second, disruptive AGR2 protomer exploits rare fluctuations in the IRE1β dimer that expose its binding site. Thus, AGR2 actively disrupts IRE1β dimers to suppress the UPR, while chaperone clients compete for AGR2 to trigger UPR signalling.

Broadening of signals from atoms at interfaces can often be a limiting factor in applying solution NMR to the structure determination of complexes. Common contributors to such problems include exchange between free and bound states and the increased molecular weight of complexes relative to the free components, but another cause that can be more difficult to deal with occurs when conformational dynamics within the interface takes place at an intermediate rate on the chemical shift timescale. In this work we show how a carefully chosen mutation in the protein HMG‐D rescued such a situation, making possible high‐resolution structure determination of its complex with a dA₂ bulge DNA ligand designed to mimic a natural DNA bend, and thereby revealing a new spatial organization of the complex.

We have developed and validated a highly specific, versatile antibody to the extracellular domain of human LGR5 (α-LGR5). α-LGR5 detects LGR5 overexpression in >90% of colorectal cancer (CRC), hepatocellular carcinoma (HCC) and pre-B-ALL tumour cells and was used to generate an Antibody-Drug Conjugate (α-LGR5-ADC), Bispecific T-cell Engager (α-LGR5-BiTE) and Chimeric Antigen Receptor (α-LGR5-CAR). α-LGR5-ADC was the most effective modality for targeting LGR5 ⁺ cancer cells in vitro and demonstrated potent anti-tumour efficacy in a murine model of human NALM6 pre-B-ALL driving tumour attrition to less than 1% of control treatment. α-LGR5-BiTE treatment was less effective in the pre-B-ALL cancer model yet promoted a twofold reduction in tumour burden. α-LGR5-CAR-T cells also showed specific and potent LGR5 ⁺ cancer cell killing in vitro and effective tumour targeting with a fourfold decrease in pre-B-ALL tumour burden relative to controls. Taken together, we show that α-LGR5 can not only be used as a research tool and a biomarker but also provides a versatile building block for a highly effective immune therapeutic portfolio targeting a range of LGR5-expressing cancer cells.

Linker histones play an essential role in chromatin packaging by facilitating compaction of the 11-nm fiber of nucleosomal “beads on a string.” The result is a heterogeneous condensed state with local properties that range from dynamic, irregular, and liquid-like to stable and regular structures (the 30-nm fiber), which in turn impact chromatin-dependent activities at a fundamental level. The properties of the condensed state depend on the type of linker histone, particularly on the highly disordered C-terminal tail, which is the most variable region of the protein, both between species, and within the various subtypes and cell-type specific variants of a given organism. We have developed an in vitro model system comprising linker histone tail and linker DNA, which although very minimal, displays surprisingly complex behavior, and is sufficient to model the known states of linker histone-condensed chromatin: disordered “fuzzy” complexes (“open” chromatin), dense liquid-like assemblies (dynamic condensates), and higher-order structures (organized 30-nm fibers). A crucial advantage of such a simple model is that it allows the study of the various condensed states by NMR, circular dichroism, and scattering methods. Moreover, it allows capture of the thermodynamics underpinning the transitions between states through calorimetry. We have leveraged this to rationalize the distinct condensing properties of linker histone subtypes and variants across species that are encoded by the amino acid content of their C-terminal tails. Three properties emerge as key to defining the condensed state: charge density, lysine/arginine ratio, and proline-free regions, and we evaluate each separately using a strategic mutagenesis approach.