Biology subjects

Redfield, C.

Publications and source records attributed to Redfield, C..

14 recordsLinked to original sources

bioRxiv2026

The centromere localization domain of kinetoplastid kinetochore protein KKT2 recognizes the free N-terminus of histone H3

Kinetochores are multiprotein complexes that drive chromosome segregation in eukaryotes. Kinetoplastids, a group of early-diverging eukaryotes that lack canonical kinetochore components, have a unique set of kinetochore proteins. How their kinetochores are assembled specifically at centromeres in the absence of a centromere-specific histone H3 variant CENP-A remains unknown. Here, we demonstrate that the centromere localization (CL) domain of KKT2 has similarities to a ZZ domain. An invariant aspartate present in histone H3-binding ZZ domains is conserved in the KKT2 CL domain. Using nuclear magnetic resonance (NMR) spectroscopy and isothermal titration calorimetry (ITC), we show that Trypanosoma brucei KKT2 CL binds the N-terminus of histone H3. Strikingly, even mono-methylation of the N-terminal amino group of histone H3 abolishes the binding. Our study raises a possibility that centromere-specific kinetochore assembly in T. brucei is ensured by abundant N-terminal methylations of histone H3 in non-centromeric regions.

cell biology↗

bioRxiv2026

DNA-induced conformational changes in SPRTN relieve its auto-inhibitory effect on protease activity

The DNA-dependent metalloprotease SPRTN has emerged as a key enzyme in the proteolysis of DNA-protein crosslinks (DPCs), thereby protecting us against genome instability, accelerated ageing, and cancer. DNA and ubiquitin chains serve as the primary activator and catalyst of SPRTN proteolysis, respectively, but how they promote SPRTN activation and activity remains incompletely understood. To address this question, we developed a highly sensitive multi-turnover fluorescence resonance energy transfer (FRET) assay to monitor SPRTN proteolysis in real time. We found that the auto-cleaved N-terminal SPRTN fragment, comprising the metalloprotease domain (MPD), zinc-binding domain (ZBD), and basic region (BR), is highly stable, enzymatically active, and retains ubiquitin-dependent activation. Interestingly, the MPD alone exhibits basal intrinsic activity that is independent of both DNA activation and ubiquitin avidity effect. We show that ZBD and MPD together exert steric regulation: ZBD maintains MPD in an autoinhibited state, while MPD largely prevents ZBD from binding DNA. BR, together with DNA, is essential to relieve ZBD-mediated inhibition of MPD. Using a site-trapping approach, we demonstrate that the ZBD-BR-DNA trinity induces an open conformation of the SPRTN N-terminus in cis, thereby releasing autoinhibition. MPD and BR together restrict the DNA-binding stoichiometry of ZBD, enabling SPRTN to function efficiently in proximity to DNA despite its low abundance in vivo. Collectively, our work overturns the long-standing dogma that SPRTN autocleavage inactivates the enzyme and reveals how DNA-induced conformational changes in SPRTN fine-tune its protease activity, providing a prerequisite for subsequent ubiquitin activation and rapid proteolysis of DPCs.

biochemistry↗

bioRxiv2026

YdbL directly modulates YdbH-YnbE bridge formation to maintain Escherichia coli outer membrane homeostasis

Gram-negative bacteria pose a threat to global healthcare mainly because their outer membrane (OM) provides an intrinsic barrier to many antimicrobials. Key to this barrier function is the asymmetric structure of the OM, with phospholipids constituting the inner leaflet and lipopolysaccharides the outer leaflet. Although the mechanism of phospholipid transport between the inner membrane (IM) and OM remains poorly understood, recent studies implicate TamB, YhdP, and YdbH as functionally redundant proteins mediating this process in Escherichia coli. Accordingly, collective loss of these three paralogs is lethal and any one of them is sufficient for growth. YdbH is anchored to the IM and its periplasmic repeating {beta}-sheet groove domain interacts with the OM lipoprotein YnbE via {beta}-strand augmentation to form an intermembrane bridge. Additionally, YnbE multimerizes, and the periplasmic protein YdbL is proposed to modulate YnbE multimerization to facilitate its stacking on the C-terminus of YdbH. Here, we demonstrate that excess YdbL specifically inhibits the function of the YdbH-YnbE complex since overexpression of ydbL causes lethality in the {Delta}yhdP {Delta}tamB double mutant but the presence of both ydbH and ynbE in trans abrogates this lethality. We resolve high-resolution structural data for YdbL and ascertain its interaction site with the YnbE C-terminal -helix, with residues mediating this interface highly conserved and critical for YdbL function. Finally, we show that YdbL is protected from degradation by the protease DegP when complexed with YnbE. Overall, our data supports a model in which YdbL ensures proper YdbH-YnbE intermembrane bridge formation by directly interacting with YnbE.

microbiology↗

bioRxiv2024

The dual ubiquitin binding mode of SPRTN secures rapid spatiotemporal proteolysis of DNA-protein crosslinks

DNA-protein crosslinks (DPCs) are endogenous and chemotherapy-induced genotoxic DNA lesions and, if not repaired, lead to embryonic lethality, neurodegeneration, premature ageing, and cancer. DPCs are heavily polyubiquitinated, and the SPRTN protease and 26S proteasome emerged as two central enzymes for DPC proteolysis. The proteasome recognises its substrates by their ubiquitination status. How SPRTN protease, an essential enzyme for DPC proteolysis, achieves specificity for DPCs still needs to be discovered. We found that the N-terminal SPRTN catalytic region (SprT) possesses a ubiquitin-binding domain named the Ubiquitin interface of SprT Domain (USD). Using multiple biochemical, biophysical, and structural approaches, we reveal that USD binds ubiquitin chains. SPRTN binding to ubiquitin chains via USD leads to [~] 67-fold higher activation of SPRTN proteolysis towards polyubiquitinated DPCs than the unmodified DPCs. This study reveals the ubiquitination of DPCs is the key signal for SPRTNs substrate specificity and rapid proteolysis.

biochemistry↗

bioRxiv2024

Engineered HMGB1 Construct with Tandem HMG B Domains that Promotes Tissue Regeneration without Potential for Deleterious Inflammation or Thrombosis

Fully reduced High Mobility Group Box 1 (HMGB1) binds CXCL12 and signals via CXCR4 when released into the extracellular space. It acts as a chemokine and transitions stem cells from quiescent G to a primed GAlert state. Cells in GAlert rapidly enter G1 in response to activating factors released by tissue injury to promote tissue repair. However, oxidative conversion of FR-HMGB1 into the disulfide form activates proinflammatory pathways via TLR-2, TLR-4 and RAGE. Peptide mapping and NMR spectroscopy identified a conserved CXCL12-binding motif within each Box and adjacent flanking regions. We decoupled the regenerative and inflammatory functions using an engineered construct (dBB12L), comprising tandem Box B domains with a flexible linker. dBB12L exhibited CXCL12 binding and accelerated repair equivalent to FR-HMGB1. Importantly, dBB12L lacked detectable RAGE binding and did not signal via TLR-2 and TLR-4, establishing it as a potential therapeutic to promote tissue repair without deleterious inflammation.

biochemistry↗

bioRxiv2024

Structural basis for carbohydrate recognition by the Gal/GalNAc lectin of Entamoeba histolytica

Intestinal amoebiasis is caused by Entamoeba histolytica, one of the deadliest human-infective parasites. Central to its pathogenicity is its binding to mucosal carbohydrates, which precedes tissue damage by trogocytosis. Carbohydrate binding is mediated by a single adhesin, the galactose/N-acetylgalactosamine (Gal/GalNAc) lectin, which is the leading vaccine candidate for amoebiasis. We present the structure of the native heterodimeric lectin, revealing an ordered core containing the light chain and the N-terminal region of the heavy chain. Structures in the presence of ligand show that the Gal/GalNAc binding site is in the light chain. An elongated arm emerges from the heavy chain, which adopts multiple positions. This arm contains the epitope for a monoclonal antibody which blocks binding to target cells, located unexpectedly away from the Gal/GalNAc binding site. This study reveals the molecular basis for sugar binding by the Entamoeba histolytica Gal/GalNAc lectin and will guide future vaccine development against amoebiasis.

microbiology↗

bioRxiv2024

The kinetoplastid kinetochore protein KKT23 acetyltransferase is a structural homolog of GCN5 that acetylates the histone H2A C-terminal tail

The kinetochore is the macromolecular protein machine that drives chromosome segregation in eukaryotes. In an evolutionarily divergent group of organisms called kinetoplastids, kinetochores are built using a unique set of proteins (KKT1-25 and KKIP1-12). KKT23 is a constitutively localised kinetochore protein containing a C-terminal acetyltransferase domain of unknown function. Here, using X-ray crystallography and NMR spectroscopy, we have determined the structure and dynamics of the KKT23 acetyltransferase domain from Trypanosoma brucei and found that it is structurally similar to the GCN5 histone acetyltransferase domain. We find that KKT23 can acetylate the C-terminal tail of histone H2A and that knockdown of KKT23 results in decreased H2A acetylation levels in T. brucei. Finally, we have determined the crystal structure of the N-terminal region of KKT23 and shown that it interacts with KKT22. Our study provides important insights into the structure and function of the unique kinetochore acetyltransferase in trypanosomes.

cell biology↗

bioRxiv2024

The transition state for coupled folding and binding of a disordered DNA binding domain resembles the unbound state

The basic zippers (bZIPs) are one of two large eukaryotic families of transcription factors whose DNA binding domains are disordered in isolation but fold into stable -helices upon target DNA binding. Here we systematically disrupt pre-existing helical propensity within the DNA binding region of the homodimeric bZIP domain of cAMP-response element binding protein (CREB) using Ala-Gly scanning and examine the impact on target binding kinetics. We find that the secondary structure of the transition state strongly resembles that of the unbound state. The closest residue to the dimerisation domain that has been examined is largely folded within both unbound and transition states; dimerisation apparently propagates additional helical propensity into the basic region. The results are consistent with electrostatically-enhanced DNA binding, followed by rapid folding from the folded zipper outwards. Interestingly, despite taking the exact experimental approach suggested for testing it, we find no evidence for disorder-mediated rate enhancement predicted by fly-casting theory.

biophysics↗

bioRxiv2022

Molecular determinants of dynamic protein-protein interactions in the functional cycle of the membrane protein DsbD

Molecular recognition is of central importance in biology. The molecular determinants shaping recognition of one protein domain by another are incompletely understood, especially in the context of the complex function of molecular machines. Here, we combine NMR experiments and molecular dynamics simulations to elucidate the determinants of recognition of the C-terminal (cDsbD) domain of the transmembrane reductant conductor DsbD by its cognate partner, the N-terminal domain of the protein (nDsbD). As part of the natural cycle of this oxidoreductase, which effectively transfers electrons from the cytoplasm to the periplasm of Gram-negative bacteria, cDsbD and nDsbD toggle between oxidised and reduced states, something that modulates the affinity of the domains for each other and prevents otherwise unproductive reactions. We find that the redox state of cDsbD determines the dissociation rate of cDsbD-nDsbD complexes. Molecular dynamics simulations demonstrate how the redox-state of the active site determines the stability of inter-domain hydrogen bonds and thus the dissociation rate. AlphaFold modelling and atomistic molecular dynamics simulations of full-length DsbD in a realistic bacterial membrane again highlights the close proximity of the periplasmic domains and the importance of tuning the strength of the interactions of the periplasmic domains to enable electron transfer to cognate periplasmic partners such as CcmG. Our AlphaFold models are consistent with in vivo functional assays of DsbD mutants, which together help to reveal for the first-time a putative binding site for thioredoxin on the cytoplasmic side of DsbD.

biophysics↗

bioRxiv2020

Structural characterisation of KKT4, an unconventional microtubule-binding kinetochore protein

The kinetochore is the macromolecular protein machinery that drives chromosome segregation by interacting with spindle microtubules. Unlike most other eukaryotes that have canonical kinetochore proteins, a group of evolutionarily divergent eukaryotes called kinetoplastids (such as Trypanosoma brucei) have a unique set of kinetochore proteins. To date, KKT4 is the only kinetoplastid kinetochore protein that is known to bind microtubules. Here we use X-ray crystallography, NMR spectroscopy, and crosslinking mass spectrometry to characterise the structure and dynamics of KKT4. We show that its microtubule-binding domain consists of a coiled-coil structure followed by a positively charged disordered tail. The crystal structure of the C-terminal BRCT domain of KKT4 reveals that it is likely a phosphorylation-dependent protein-protein interaction domain. The BRCT domain interacts with the N-terminal region of the KKT4 microtubule-binding domain and with a phosphopeptide derived from KKT8. Finally, we show that KKT4 binds DNA with high affinity. Taken together, these results provide the first structural insights into the unconventional kinetoplastid kinetochore protein KKT4.

cell biology↗

bioRxiv2019

Pal stabilises the bacterial outer membrane during constriction by a mobilisation-and-capture mechanism

Coordination of outer membrane constriction with septation is critical to faithful division in Gram-negative bacteria and vital to the barrier function of the membrane. Recent studies suggest this coordination is through the active accumulation of the peptidoglycan-binding outer membrane lipoprotein Pal at division sites by the Tol system, but the mechanism is unknown. Here, we show that Pal accumulation at Escherichia coli division sites is a consequence of three key functions of the Tol system. First, Tol mobilises Pal molecules in dividing cells, which otherwise diffuse very slowly due to their binding of the cell wall. Second, Tol actively captures mobilised Pal molecules and deposits them at the division septum. Third, the active capture mechanism is analogous to that used by the inner membrane protein TonB to dislodge the plug domains of outer membrane TonB-dependent nutrient transporters. We conclude that outer membrane constriction is coordinated with cell division by active mobilisation-and-capture of Pal at division septa by the Tol system.

microbiology↗

bioRxiv2019

Local frustration determines loop opening in protein-protein association

Local structural frustration, the existence of mutually exclusive competing interactions, may explain why some proteins are dynamic while others are rigid. Frustration is thought to underpin biomolecular recognition and the flexibility of protein binding sites. Here we show how a small chemical modification, the oxidation of two cysteine thiols to a disulfide bond, during the catalytic cycle of the N-terminal domain of the key bacterial oxidoreductase DsbD (nDsbD), introduces frustration ultimately influencing protein function. In oxidized nDsbD, local frustration disrupts the packing of the protective cap-loop region against the active site allowing loop opening. By contrast, in reduced nDsbD the cap loop is rigid, always protecting the active-site thiols from the oxidizing environment of the periplasm. Our results point towards an intricate coupling between the dynamics of the active-site cysteines and of the cap loop which modulates the association reactions of nDsbD with its partners resulting in optimized protein function.

biophysics↗

bioRxiv2019

Conformational flexibility of GRASP protein and its constituent PDZ subdomains reveals structural basis of its promiscuous interactome

The Golgi complex is a central component of the secretory pathway, responsible for several critical cellular functions in eukaryotes. The complex is organized by the Golgi matrix, which includes the Golgi Reassembly and Stacking Proteins (GRASPs), which participate in cisternae stacking and lateral linkage in vertebrates. GRASPs also have critical roles in other processes, with an unusual ability to interact with several different protein binding partners. The conserved N-terminus of the GRASP family includes two PDZ domains. Previous crystallographic studies of orthologues suggest that PDZ1 and PDZ2 have similar conformations and secondary structure content, however PDZ1 alone mediates nearly all the interactions between GRASPs and their binding partners. In this work, NMR, Synchrotron-Radiation Circular Dichroism and Molecular Dynamics were used to examine the structure, flexibility and stability of the two constituent PDZ domains. GRASP PDZs are structured in an unusual {beta}31{beta}4{beta}52{beta}6{beta}1{beta}2 secondary structural arrangement and NMR data indicates that the PDZ1 binding pocket is formed by a stable {beta}2-strand and a more flexible and unstable 2-helix, suggesting an explanation for the higher PDZ1 promiscuity. The conformational free energy profiles of the two PDZ domains were calculated using Molecular Dynamics simulations. The data suggest that, after binding, the protein partner significantly reduces the conformational space that GRASPs can access by stabilizing one particular conformation, in a partner-dependent fashion. The structural flexibility of PDZ1, modulated by PDZ2, and the coupled, coordinated movement between the two PDZs enable GRASPs to interact with multiple partners, allowing them to function as promiscuous, multitasking proteins.\n\nSignificance StatementGolgi Reassembly and Stacking Proteins (GRASPs) play pivotal roles in the maintenance of Golgi structure as well as in unconventional protein secretion. Their broad network of interactions is mainly sustained by the two-PDZ domains located in the N-terminal portion of the protein. The asymmetry of the PDZ domains in terms of number and diversity of interacting partners has been long recognized, but the molecular determinants of that asymmetry remains largely unknown. The biophysical data presented here provide a firm basis for understanding why PDZ1 behaves differently to PDZ2 in solution, despite their similar 3D structures. Furthermore, we propose that PDZ2 assist ligand binding to PDZ1, by means of conformational stabilization.

biophysics↗

bioRxiv2017

Derivation and Validation of a Record Linkage Algorithm between EMS and the Emergency Department

BackgroundLinking EMS electronic patient care reports (ePCRs) to ED records can provide clinicians access to vital information that can alter management. It can also create rich databases for research and quality improvement. Unfortunately, previous attempts at ePCR - ED record linkage have had limited success.\n\nObjectiveTo derive and validate an automated record linkage algorithm between EMS ePCRs and ED records using supervised machine learning.\n\nMethodsAll consecutive ePCRs from a single EMS provider between June 2013 and June 2015 were included. A primary reviewer matched ePCRs to a list of ED patients to create a gold standard. Age, gender, last name, first name, social security number (SSN), and date of birth (DOB) were extracted. Data was randomly split into 80%/20% training and test data sets. We derived missing indicators, identical indicators, edit distances, and percent differences. A multivariate logistic regression model was trained using 5k fold cross-validation, using label k-fold, L2 regularization, and class re-weighting.\n\nResultsA total of 14,032 ePCRs were included in the study. Inter-rater reliability between the primary and secondary reviewer had a Kappa of 0.9. The algorithm had a sensitivity of 99.4%, a PPV of 99.9% and AUC of 0.99 in both the training and test sets. DOB match had the highest odd ratio of 16.9, followed by last name match (10.6). SSN match had an odds ratio of 3.8.\n\nConclusionsWe were able to successfully derive and validate a probabilistic record linkage algorithm from a single EMS ePCR provider to our hospital EMR.

bioinformatics↗