Biology subjects

Williams, H. D.

Publications and source records attributed to Williams, H. D..

5 recordsLinked to original sources

bioRxiv2025

A Flexible Quadruple-Stranded Helicate Demonstrates a Strong Binding Preference for DNA Three-Way Junctions by Induced-Fit

Nucleic acid junctions are key to many biological functions from recombination and repair to viral NA insertion, and are an attractive, functional biomolecular target. We describe a quadruple-stranded di-platinum helicate that binds both three-way (3WJ) and four-way DNA junctions (4WJ). This allows us to probe the relative importance of size and shape in junction-binder design. Despite the helicates tetragonal symmetry/shape being compatible with the 4WJ, microscale thermophoresis (MST), isothermal calorimetry (ITC) and gel electrophoresis competition experiments demonstrate that this metallo-supramolecule displays a stronger affinity for 3WJs (Kd = 12 nM) than for 4WJs (Kd > 4 {micro}M) and other DNA structures. The experimental findings are supported by molecular dynamics simulations which reveal the critical role of size. Whilst the open form of the 4WJ is promoted when the helicate is in the cavity, the helicates small size means it is unable to maintain {pi} contacts with all four junction base-pairs simultaneously. Although the helicate is slightly too large for the smaller 3WJ cavity, simulations and experiments show that it can open up the cavity (increasing the junctions hydrodynamic radius) by disrupting a base-pair. The flexible helicate also responds to the cavity upon binding by favouring one enantiomer and allowing the helicate to adopt a stable final structure inside the 3WJ that is an induced-fit of the two dynamic structures (supramolecule and DNA). This contrasts with previous lock-and-key examples of junction recognition and opens up new possibilities for how to design DNA and RNA junction-binding compounds. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=72 SRC="FIGDIR/small/671622v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@175cabborg.highwire.dtl.DTLVardef@50f114org.highwire.dtl.DTLVardef@372e1org.highwire.dtl.DTLVardef@6b2960_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2023

Laser Desorption Rapid Evaporative Ionisation Mass Spectrometry (LD-REIMS) demonstrates a direct impact of hypochlorous acid stress on PQS-mediated quorum sensing in Pseudomonas aeruginosa

To establish infections in human hosts, Pseudomonas aeruginosa must overcome innate immune generated oxidative stress, such as the hypochlorous acid (HOCl) produced by neutrophils. We set out to find specific biomarkers of oxidative stress through the development of a protocol for the metabolic profiling of P. aeruginosa cultures grown in the presence of different oxidants using a novel ionisation technique for mass spectrometry, laser desorption rapid evaporative ionisation mass spectrometry (LD-REIMS). We demonstrated the ability of LD-REIMS to classify samples as untreated or treated with a specific oxidant with 100 % accuracy and identified a panel of 54 metabolites with significantly altered concentrations after exposure to one or more of the oxidants. Key metabolic changes were conserved in P. aeruginosa clinical strains isolated from patients with cystic fibrosis lung infections. These data demonstrated that HOCl stress impacted the Pseudomonas Quinolone Signal (PQS) quorum sensing system. Ten 2-alkyl-4-quinolones (AHQs) associated with the PQS system were significantly lower in concentration in HOCl-stressed P. aeruginosa cultures, including 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS), the most active signal molecule of the PQS system. The PQS system regulates the production of virulence factors, including pyocyanin and elastase, and their levels were markedly affected by HOCl stress. No pyocyanin was detectable and elastase concentrations were significantly reduced in cultures grown with sub-lethal concentrations of HOCl, suggesting that this neutrophil-derived oxidant may disrupt the ability of P. aeruginosa to establish infections through interference with production of PQS-associated virulence factors. IMPORTANCEThis work demonstrates that a high-throughput ambient ionisation mass spectrometry method can be used successfully to study a bacterial stress response. Its application to the opportunistic pathogen Pseudomonas aeruginosa led to the identification of specific oxidative stress biomarkers, and demonstrated that hypochlorous acid, an oxidant specifically produced by human neutrophils during infection, affects quorum sensing and markedly reduces production of the virulence factors pyocyanin and elastase in this bacterium. This approach has the potential to be widely applicable to the characterisation of the stress responses of bacteria.

microbiology↗

bioRxiv2023

Organometallic Pillarplexes that bind DNA 4-way Holliday Junctions and Forks.

Holliday 4-way junctions are key to important biological DNA processes (insertion, recombination and repair) and are dynamic structures which adopt either open or closed conformations, with the open conformation being the biologically active form. Tetracationic metallo-supramolecular pillarplexes display aryl faces about a cylindrical core giving them an ideal structure to interact with the central cavities of open DNA junctions. Combining experimental studies and MD simulations we show that an Au pillarplex can bind DNA 4-way junctions (Holliday junctions) in their open form, a binding mode not accessed by synthetic agents before. The Au pillarplexes can bind designed 3-way junctions too but their large size leads them to open up and expand that junction, disrupting the base pairing which manifests in an increase in hydrodynamic size and a lower junction thermal stability. At high loading they re-arrange both 4-way and 3-way junctions into Y-shaped DNA forks to increase the available junction-like binding sites. The structurally related Ag pillarplexes show similar DNA junction binding behaviour, but a lower solution stability. This pillarplex binding contrasts with (but complements) that of the metallo-supramolecular cylinders, which prefer 3-way junctions and we show can rearrange 4-way junctions into 3-way junction structures. The pillarplexes ability to bind open 4-way junctions creates exciting possibilities to modulate and switch such structures in biology, as well as in synthetic nucleic acid nanostructures where they are key interconnecting components. Studies in human cells, confirm that the pillarplexes do reach the nucleus, with antiproliferative activity at levels similar to those of cisplatin. The findings provide a new roadmap for targeting higher order junction structures using a metallo-supramolecular approach, as well as expanding the toolbox available to design bioactive junction-binders into organometallic chemistry. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=188 SRC="FIGDIR/small/522759v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@10fac36org.highwire.dtl.DTLVardef@1f32f9forg.highwire.dtl.DTLVardef@bb8d72org.highwire.dtl.DTLVardef@1433de5_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

A universal stress protein acts as a metabolic rheostat controlling carbon flux in mycobacteria

Universal stress proteins (USPs) are ubiquitous amongst prokaryotes and have empirically determined roles in adaptation to stress, while their specific biochemical function in cellular processes is opaque. The most frequently encountered USP is a small ~15kDa protein comprised of a single domain (PF00582). In Mycobacterium tuberculosis Rv1636 is the sole single domain protein amongst its 10 USPs. We were unable to delete rv1636 from M. tuberculosis, supporting previous data that it is essential for growth. We deleted its orthologue, MS3811, from M. smegmatis, and in exponential phase competition experiments we observed that the advantage switched between wildtype and the mutant depending on the growth environment. In rich medium the wildtype had the competitive advantage, but in a defined medium with a single carbon source the competitive advantage switched, with the mutant rapidly taking over the culture. We hypothesised the USP is regulating metabolic flux, with its deletion leading to increased flux and more rapid turn-over of central-carbon metabolites. We tested this by performing 13C-stable isotope tracing, using [U-13C6] glucose as sole carbon source for growth of the parental and deletion strains. The findings were unequivocal; deletion of the USP led to an increase in label incorporation in central carbon metabolites and a profound change in the isotopologue distribution. Furthermore, in vivo protein-crosslinking provided evidence that Rv1636 interacts directly with key central metabolic enzymes, including the glycolytic enzymes pyruvate kinase, pyruvate dehydrogenase, pyruvate synthase. We propose the mycobacterial single domain USP acts as a metabolic rheostat to regulate central carbon metabolism. ImportanceThere are more than 62,000 universal stress proteins (USPs) found mostly in prokaryotes, but very few examples where the role of a USP is unequivocally linked to a specific biological or biochemical function. For the first time we were able to assign a role in metabolic regulation to a bacterial single domain universal stress protein. We demonstrate a mycobacterial USP regulates flux through central catabolic pathways, and our evidence, based on 13C-stable isotope tracing, indicates that the USP acts as a brake on carbon flux, and we assign to it a role as a metabolic rheostat.

microbiology↗

bioRxiv2020

Response of Pseudomonas aeruginosa to the innate immune system-derived oxidants hypochlorous acid and hypothiocyanous acid

Pseudomonas aeruginosa is a significant nosocomial pathogen and associated with lung infections in cystic fibrosis (CF). Once established, P. aeruginosa infections persist and are rarely eradicated despite the host immune cells producing antimicrobial oxidants, including hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN). There is limited knowledge as to how P. aeruginosa senses, responds to, and survives attack from HOCl and HOSCN, and the contribution of such responses to its success as a CF pathogen. We investigated the P. aeruginosa response to these oxidants by screening 707 transposon mutants, with mutations in regulatory genes, for altered growth following HOCl exposure. We identified regulators involved in antibiotic resistance, methionine biosynthesis and catabolite repression, and PA14_07340, the homologue of the Escherichia coli HOCl-sensor RclR (30% identical), that were required for HOCl survival. We have shown that RclR (PA14_07340) protects specifically against HOCl and HOSCN stress, and responds to both oxidants by upregulating expression of a putative peroxiredoxin, rclX (PA14_07355). While there was specificity in the transcriptional response to HOCl (231 genes upregulated) and HOSCN (105 genes upregulated) there was considerable overlap, with 74 genes upregulated by both oxidants. These included genes encoding the type III secretion system (T3SS), sulphur and taurine transport, and the MexEF-OprN efflux pump. RclR coordinated the transcriptional response to HOCl and HOSCN, including upregulation of pyocyanin biosynthesis genes, and in response to HOSCN alone RclR downregulated chaperone genes. These data indicate that the P. aeruginosa response to HOCl and HOSCN is multifaceted, with RclR playing an essential role.

microbiology↗