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Journal articleSgueglia G, Vrettas MD, Chino M, et al., 2023,
MetalHawk: Enhanced Classification of Metal Coordination Geometries by Artificial Neural Networks
, JOURNAL OF CHEMICAL INFORMATION AND MODELING, Vol: 64, Pages: 2356-2367, ISSN: 1549-9596 -
Journal articleHutchison CDM, Baxter JM, Fitzpatrick A, et al., 2023,
Optical control of ultrafast structural dynamics in a fluorescent protein
, NATURE CHEMISTRY, ISSN: 1755-4330 -
Journal articleMcKenna S, Aylward F, Miliara X, et al., 2023,
The protease associated (PA) domain in ScpA from Streptococcus pyogenes plays a role in substrate recruitment
, BBA: Proteins and Proteomics, Vol: 1871, Pages: 1-11, ISSN: 1570-9639Annually, over 18 million disease cases and half a million deaths worldwide are estimated to be caused by Group A Streptococcus. ScpA (or C5a peptidase) is a well characterised member of the cell enveleope protease family, which possess a S8 subtilisin-like catalytic domain and a shared multi-domain architecture. ScpA cleaves complement factors C5a and C3a, impairing the function of these critical anaphylatoxins and disrupts complement-mediated innate immunity. Although the high resolution structure of ScpA is known, the details of how it recognises its substrate are only just emerging. Previous studies have identified a distant exosite on the 2nd fibronectin domain that plays an important role in recruitment via an interaction with the substrate core. Here, using a combination of solution NMR spectroscopy, mutagenesis with functional assays and computational approaches we identify a second exosite within the protease-associated (PA) domain. We propose a model in which the PA domain assists optimal delivery of the substrate's C terminus to the active site for cleavage.
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Journal articleSchloesser L, Sachse C, Low HH, et al., 2023,
Conserved structures of ESCRT-III superfamily members across domains of life
, TRENDS IN BIOCHEMICAL SCIENCES, Vol: 48, Pages: 993-1004, ISSN: 0968-0004 -
Journal articleThabet MA, Penadés JR, Haag AF, 2023,
The ClpX protease is essential for inactivating the CI master repressor and completing prophage induction in Staphylococcus aureus
, Nature Communications, Vol: 14, ISSN: 2041-1723Bacteriophages (phages) are the most abundant biological entities on Earth, exerting a significant influence on the dissemination of bacterial virulence, pathogenicity, and antimicrobial resistance. Temperate phages integrate into the bacterial chromosome in a dormant state through intricate regulatory mechanisms. These mechanisms repress lytic genes while facilitating the expression of integrase and the CI master repressor. Upon bacterial SOS response activation, the CI repressor undergoes auto-cleavage, producing two fragments with the N-terminal domain (NTD) retaining significant DNA-binding ability. The process of relieving CI NTD repression, essential for prophage induction, remains unknown. Here we show a specific interaction between the ClpX protease and CI NTD repressor fragment of phages Ф11 and 80α in Staphylococcus aureus. This interaction is necessary and sufficient for prophage activation after SOS-mediated CI auto-cleavage, defining the final stage in the prophage induction cascade. Our findings unveil unexpected roles of bacterial protease ClpX in phage biology.
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Journal articleSammon D, Krueger A, Busse-Wicher M, et al., 2023,
Molecular mechanism of decision-making in glycosaminoglycan biosynthesis
, Nature Communications, Vol: 14, ISSN: 2041-1723Two major glycosaminoglycan types, heparan sulfate (HS) and chondroitin sulfate (CS), control many aspects of development and physiology in a type-specific manner. HS and CS are attached to core proteins via a common linker tetrasaccharide, but differ in their polymer backbones. How core proteins are specifically modified with HS or CS has been an enduring mystery. By reconstituting glycosaminoglycan biosynthesis in vitro, we establish that the CS-initiating N-acetylgalactosaminyltransferase CSGALNACT2 modifies all glycopeptide substrates equally, whereas the HS-initiating N-acetylglucosaminyltransferase EXTL3 is selective. Structure-function analysis reveals that acidic residues in the glycopeptide substrate and a basic exosite in EXTL3 are critical for specifying HS biosynthesis. Linker phosphorylation by the xylose kinase FAM20B accelerates linker synthesis and initiation of both HS and CS, but has no effect on the subsequent polymerisation of the backbone. Our results demonstrate that modification with CS occurs by default and must be overridden by EXTL3 to produce HS.
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Journal articleEl Omari K, Duman R, Mykhaylyk V, et al., 2023,
Experimental phasing opportunities for macromolecular crystallography at very long wavelengths
, COMMUNICATIONS CHEMISTRY, Vol: 6, ISSN: 2399-3669 -
Journal articleCosta TRD, Patkowski JB, Mace K, et al., 2023,
Structural and functional diversity of type IV secretion systems
, Nature Reviews Microbiology, Vol: 22, Pages: 170-185, ISSN: 1740-1526Considerable progress has been made in recent years in the structural and molecular biology of type IV secretion systems in Gram-negative bacteria. The latest advances have substantially improved our understanding of the mechanisms underlying the recruitment and delivery of DNA and protein substrates to the extracellular environment or target cells. In this Review, we aim to summarize these exciting structural and molecular biology findings and to discuss their functional implications for substrate recognition, recruitment and translocation, as well as the biogenesis of extracellular pili. We also describe adaptations necessary for deploying a breadth of processes, such as bacterial survival, host–pathogen interactions and biotic and abiotic adhesion. We highlight the functional and structural diversity that allows this extremely versatile secretion superfamily to function under different environmental conditions and in different bacterial species. Additionally, we emphasize the importance of further understanding the mechanism of type IV secretion, which will support us in combating antimicrobial resistance and treating type IV secretion system-related infections.
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Journal articleJonsson R, Bjorling A, Midtgaard SR, et al., 2023,
Aggregative adherence fimbriae form compact structures as seen by SAXS
, SCIENTIFIC REPORTS, Vol: 13, ISSN: 2045-2322 -
Journal articleThurston TLM, Holden DW, 2023,
The Salmonella Typhi SPI-2 injectisome enigma
, MICROBIOLOGY-SGM, Vol: 169, ISSN: 1350-0872 -
Journal articleRajoub N, Gerard CJJ, Pantuso E, et al., 2023,
A workflow for the development of template-assisted membrane crystallization downstream processing for monoclonal antibody purification
, Nature Protocols, Vol: 18, Pages: 2998-3049, ISSN: 1750-2799Monoclonal antibodies (mAbs) are commonly used biologic drugs for the treatment of diseases such as rheumatoid arthritis, multiple sclerosis, COVID-19 and various cancers. They are produced in Chinese hamster ovary cell lines and are purified via a number of complex and expensive chromatography-based steps, operated in batch mode, that rely heavily on protein A resin. The major drawback of conventional procedures is the high cost of the adsorption media and the extensive use of chemicals for the regeneration of the chromatographic columns, with an environmental cost. We have shown that conventional protein A chromatography can be replaced with a single crystallization step and gram-scale production can be achieved in continuous flow using the template-assisted membrane crystallization process. The templates are embedded in a membrane (e.g., porous polyvinylidene fluoride with a layer of polymerized polyvinyl alcohol) and serve as nucleants for crystallization. mAbs are flexible proteins that are difficult to crystallize, so it can be challenging to determine the optimal conditions for crystallization. The objective of this protocol is to establish a systematic and flexible approach for the design of a robust, economic and sustainable mAb purification platform to replace at least the protein A affinity stage in traditional chromatography-based purification platforms. The procedure provides details on how to establish the optimal parameters for separation (crystallization conditions, choice of templates, choice of membrane) and advice on analytical and characterization methods.
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Journal articleWiggins BG, Wang Y-F, Burke A, et al., 2023,
Endothelial sensing of AHR ligands regulates intestinal homeostasis
, Nature, Vol: 621, Pages: 821-829, ISSN: 0028-0836Endothelial cells (ECs) line the blood and lymphatic vasculature, and act as an essential physical barrier, control nutrient transport, facilitate tissue immunosurveillance, and coordinate angiogenesis/ lymphangiogenesis1,2. In the intestine, dietary and microbial cues are particularly important in the regulation of organ homeostasis. However, whether enteric ECs actively sense and integrate such signals is currently unknown. Here, we show that the aryl hydrocarbon receptor (AHR) acts as a critical node for EC-sensing of dietary metabolites in adult mice and human primary ECs. We first established a comprehensive single-cell endothelial atlas of the mouse small intestine, uncovering the cellular complexity and functional heterogeneity of blood and lymphatic ECs. Analyses of AHR mediated responses at single-cell resolution identified tissue-protective transcriptional signatures and regulatory networks promoting cellular quiescence and vascular normalcy at steady state. Endothelial AHR-deficiency in adult mice resulted in dysregulated inflammatory responses, and the initiation of proliferative pathways. Furthermore, endothelial sensing of dietary AHR ligands was required for optimal protection against enteric infection. In human ECs, AHR signalling promoted quiescence and restrained activation by inflammatory mediators. Together, our data provide a comprehensive dissection of the impact of environmental sensing across the spectrum of enteric endothelia, demonstrating that endothelial AHR signalling integrates dietary cues to maintain tissue homeostasis by promoting EC quiescence and vascular normalcy.
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Journal articleBoussac A, Sugiura M, Nakamura M, et al., 2023,
Absorption changes in Photosystem II in the Soret band region upon the formation of the chlorophyll cation radical [P<sub>D1</sub>P<sub>D2</sub>]<SUP>+</SUP>
, PHOTOSYNTHESIS RESEARCH, ISSN: 0166-8595 -
Journal articleFrankel G, 2023,
Plasmids pick a bacterial partner before committing to conjugation (vol 51, pg 8925, 2023)
, NUCLEIC ACIDS RESEARCH, Vol: 51, Pages: 10812-10812, ISSN: 0305-1048 -
Journal articleFrankel G, David S, Low WW, et al., 2023,
Plasmids pick a bacterial partner before committing to conjugation
, NUCLEIC ACIDS RESEARCH, Vol: 51, Pages: 8925-8933, ISSN: 0305-1048- Cite
- Citations: 15
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Journal articleLow H, Tassinari M, Filloux A, et al., 2023,
Assembly mechanism of a Tad secretion system secretin-pilotin complex
, Nature Communications, Vol: 14, ISSN: 2041-1723The bacterial Tight adherence Secretion System (TadSS) assembles surface pili that drive cell adherence, biofilm formation and bacterial predation. The structure and mechanism of the TadSS is mostly unknown. This includes characterisation of the outer membrane secretin through which the pilus is channelled and recruitment of its pilotin. Here we investigate RcpA and TadD lipoprotein from Pseudomonas aeruginosa. Light microscopy reveals RcpA colocalising with TadD in P. aeruginosa and when heterologously expressed in Escherichia coli. We use cryogenic electron microscopy to determine how RcpA and TadD assemble a secretin channel with C13 and C14 symmetries. Despite low sequence homology, we show that TadD shares a similar fold to the type 4 pilus system pilotin PilF. We establish that the C-terminal four residues of RcpA bind TadD - an interaction essential for secretin formation. The binding mechanism between RcpA and TadD appears distinct from known secretin-pilotin pairings in other secretion systems.
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Journal articleGovada L, Chayen NE, 2023,
Crystallisation and characterisation of muscle proteins: a mini-review
, Journal of Muscle Research and Cell Motility, Vol: 44, Pages: 209-215, ISSN: 0142-4319The techniques of X-ray protein crystallography, NMR and high-resolution cryo-electron microscopy have all been used to determine the high-resolution structure of proteins. The most-commonly used method, however, remains X-ray crystallography but it does rely heavily on the production of suitable crystals. Indeed, the production of diffraction quality crystals remains the rate-limiting step for most protein systems. This mini-review highlights the crystallisation trials that used existing and newly developed crystallisation methods on two muscle protein targets - the actin binding domain (ABD) of α-actinin and the C0-C1 domain of human cardiac myosin binding protein C (cMyBP-C). Furthermore, using heterogenous nucleating agents the crystallisation of the C1 domain of cMyBP-C was successfully achieved in house along with preliminary actin binding studies using electron microscopy and co-sedimentation assays .
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Journal articleLink FJ, Errington E, Verma V, et al., 2023,
The role of mixing and surface hydrophobicity on the operation of a continuous tubular slug flow crystalliser for lysozyme
, Chemical Engineering Journal, Vol: 471, Pages: 1-13, ISSN: 1385-8947Continuous crystallisation is currently seen as a more economic and efficient alternative compared to standard techniques for the purification of biopharmaceutical proteins. Despite being promising as continuous crystalliser platforms, tubular slug flow crystallisers pose their challenges due to the tendency of proteins to accumulate at solid surfaces, to form amorphous precipitates at high protein concentrations and to undergo slow diffusive mass transport. This work investigates the effect of equipment surface chemistry and physical mixing on the design and operability of a tubular slug flow crystalliser for lysozyme under laminar flow conditions (Re ∼ 1). Firstly, glass tubes with different surface functional groups, –OH or –CH3, with water contact angles between 9° and 99°, were investigated. CH3 surfaces resulted in a up to 33% delayed onset of nucleation, demonstrating lower heterogeneous nucleation rates, and therefore are better suited to prevent fouling. However, a surface chemistry-independent deposition of lysozyme was found, altering the water contact angle by up to 56°, resulting in an unstable slug flow and a reduction of the onset of nucleation by up to 11.5-fold. To achieve a stable slug flow and controlled nucleation, the surface functional groups were recovered by implementing surface-specific cleaning protocols comprising NaOH or liquid detergent. To overcome poor mixing and amorphous precipitation, a two-step mixing approach, consisting of an intermediate mixing step was developed. This novel mixing approach reduced the mixing time from > 10 min to < 10 s, allowing the achievement of an instantaneous homogeneous solution under laminar flow conditions. Overall, the findings of this study are therefore of crucial relevance to the future design and operation of tubular slug flow crystallisers as purification platforms for biopharmaceutical proteins.
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Journal articleYates LA, Zhang X, 2023,
Phosphoregulation of the checkpoint kinase Mec1ATR
, DNA REPAIR, Vol: 129, ISSN: 1568-7864 -
Journal articleRamlaul K, Feng Z, Canavan C, et al., 2023,
A 3D-printed flow-cell for on-grid purification of electron microscopy samples directly from lysate
, Journal of Structural Biology, Vol: 215, Pages: 1-12, ISSN: 1047-8477While recent advances in cryo-EM, coupled with single particle analysis, have thepotential to allow structure determination in a near-native state from vanishingly few individualparticles, this vision has yet to be realised in practise. Requirements for particle numbers thatcurrently far exceed the theoretical lower limits, challenges with the practicalities of achievinghigh concentrations for difficult-to-produce samples, and inadequate sample-dependent imagingconditions, all result in significant bottlenecks preventing routine structure determination usingcryo-EM. Therefore, considerable efforts are being made to circumvent these bottlenecks bydeveloping affinity purification of samples on-grid; at once obviating the need to produce largeamounts of protein, as well as more directly controlling the variable, and sample-dependent,process of grid preparation.In this proof-of-concept study, we demonstrate a further practical step towards thisparadigm, developing a 3D-printable flow-cell device to allow on-grid affinity purification fromraw inputs such as whole cell lysates, using graphene oxide-based affinity grids. Our flow-celldevice can be interfaced directly with routinely-used laboratory equipment such as liquidchromatographs, or peristaltic pumps, fitted with standard chromatographic (1/16”) connectors,and can be used to allow binding of samples to affinity grids in a controlled environment priorto the extensive washing required to remove impurities. Furthermore, by designing a devicewhich can be 3D printed and coupled to routinely used laboratory equipment, we hope toincrease the accessibility of the techniques presented herein to researchers working towardssingle-particle macromolecular structures.
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Journal articleHuang X, Torre I, Chiappi M, et al., 2023,
Cryo-electron tomography of intact cardiac muscle reveals myosin binding protein-C linking myosin and actin filaments
, JOURNAL OF MUSCLE RESEARCH AND CELL MOTILITY, Vol: 44, Pages: 165-178, ISSN: 0142-4319- Cite
- Citations: 13
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Conference paperBubeck D, 2023,
Controlling the membrane attack complex
, Publisher: ELSEVIER GMBH, Pages: 1-1, ISSN: 0171-2985 -
Journal articleIbarra-Chavez R, Reboud J, Penades JR, et al., 2023,
Phage-inducible chromosomal islands as a diagnostic platform to capture and detect bacterial pathogens
, Advanced Science, Vol: 10, ISSN: 2198-3844Phage-inducible chromosomal islands (PICIs) are a family of phage satellites that hijack phage components to facilitate their mobility and spread. Recently, these genetic constructs are repurposed as antibacterial drones, enabling a new toolbox for unorthodox applications in biotechnology. To illustrate a new suite of functions, the authors have developed a user-friendly diagnostic system, based upon PICI transduction to selectively enrich bacteria, allowing the detection and sequential recovery of Escherichia coli and Staphylococcus aureus. The system enables high transfer rates and sensitivities in comparison with phages, with detection down to ≈50 CFU mL−1. In contrast to conventional detection strategies, which often rely on nucleic acid molecular assays, and cannot differentiate between dead and live organisms, this approach enables visual sensing of viable pathogens only, through the expression of a reporter gene encoded in the PICI. The approach extends diagnostic sensing mechanisms beyond cell-free synthetic biology strategies, enabling new synthetic biology/biosensing toolkits.
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Journal articleVarshitha G, Othman A, Colin B, et al., 2023,
Determining the hydrophobicity index of protected amino acids and common protecting groups
, Separations, Vol: 10, Pages: 1-11, ISSN: 2297-8739Peptides are in great demand in the pharmaceutical arena and a majority of these peptides contain 20 or more amino acids. They are infrequently synthesised using the fragment condensation approach. A key limitation in adopting this approach more commonly is that protected peptide fragments with high purity are often required prior to the final condensation steps. It is hypothesized that understanding the hydrophobic nature of the protected amino acids will assist with designing optimal fragment purification processes when needed. Whilst a myriad of hydrophobicity indices are reported in the literature for unprotected amino acids, the literature lacks any data regarding the protected amino acids which form the key precursor for the fragment condensation task. In this current study, hydrophobicity indices for protected amino acids with common α-amino and sidechain protecting groups were experimentally determined. Different positions for each amino acid within the peptide chain were considered, namely at the C-terminal and N-terminal as well as internal positions. These data give deep insights on the hydrophobicity of each amino acid with respect to its position in the peptide chain. The data acquired in this research facilitated the prediction of the retention time of protected peptide fragments with an uncertainty of less than ±1.5%.
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Journal articleTeh WK, Ding Y, Gubellini F, et al., 2023,
Characterization of TelE, a T7SS LXG effector exhibiting a conserved C-terminal glycine zipper motif required for toxicity
, Microbiology Spectrum, Vol: 11, ISSN: 2165-0497Streptococcus gallolyticus subsp. gallolyticus (SGG) is an opportunistic bacterial pathogen strongly associated with colorectal cancer. Here, through comparative genomics analysis, we demonstrated that the genetic locus encoding the type VIIb secretion system (T7SSb) machinery is uniquely present in SGG in two different arrangements. SGG UCN34 carrying the most prevalent T7SSb genetic arrangement was chosen as the reference strain. To identify the effectors secreted by this secretion system, we inactivated the essC gene encoding the motor of this machinery. A comparison of the proteins secreted by UCN34 wild type and its isogenic ΔessC mutant revealed six T7SSb effector proteins, including the expected WXG effector EsxA and three LXG-containing proteins. In this work, we characterized an LXG-family toxin named herein TelE promoting the loss of membrane integrity. Seven homologs of TelE harboring a conserved glycine zipper motif at the C terminus were identified in different SGG isolates. Scanning mutagenesis of this motif showed that the glycine residue at position 470 was crucial for TelE membrane destabilization activity. TelE activity was antagonized by a small protein TipE belonging to the DUF5085 family. Overall, we report herein a unique SGG T7SSb effector exhibiting a toxic activity against nonimmune bacteria.
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Journal articleBelan O, Greenhough L, Kuhlen L, et al., 2023,
Visualization of direct and diffusion-assisted RAD51 nucleation by full-length human BRCA2 protein
, Molecular Cell, Vol: 83, Pages: 2925-2940.e8, ISSN: 1097-2765Homologous recombination (HR) is essential for error-free repair of DNA double-strand breaks, perturbed replication forks (RFs), and post-replicative single-stranded DNA (ssDNA) gaps. To initiate HR, the recombination mediator and tumor suppressor protein BRCA2 facilitates nucleation of RAD51 on ssDNA prior to stimulation of RAD51 filament growth by RAD51 paralogs. Although ssDNA binding by BRCA2 has been implicated in RAD51 nucleation, the function of double-stranded DNA (dsDNA) binding by BRCA2 remains unclear. Here, we exploit single-molecule (SM) imaging to visualize BRCA2-mediated RAD51 nucleation in real time using purified proteins. We report that BRCA2 nucleates and stabilizes RAD51 on ssDNA either directly or through an unappreciated diffusion-assisted delivery mechanism involving binding to and sliding along dsDNA, which requires the cooperative action of multiple dsDNA-binding modules in BRCA2. Collectively, our work reveals two distinct mechanisms of BRCA2-dependent RAD51 loading onto ssDNA, which we propose are critical for its diverse functions in maintaining genome stability and cancer suppression.
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Journal articleChan DTC, Baldwin GS, Bernstein HC, 2023,
Revealing the Host-Dependent Nature of an Engineered Genetic Inverter in Concordance with Physiology
, BIODESIGN RESEARCH, Vol: 5 -
Journal articleZhao Z, Vercellino I, Knoppová J, et al., 2023,
The Ycf48 accessory factor occupies the site of the oxygen-evolving manganese cluster during photosystem II biogenesis
, Nature Communications, Vol: 14, Pages: 1-11, ISSN: 2041-1723Robust oxygenic photosynthesis requires a suite of accessory factors to ensure efficient assembly and repair of the oxygen-evolving photosystem two (PSII) complex. The highly conserved Ycf48 assembly factor binds to the newly synthesized D1 reaction center polypeptide and promotes the initial steps of PSII assembly, but its binding site is unclear. Here we have used cryo-electron microscopy to determine the structure of a cyanobacterial PSII D1/D2 reaction center assembly complex with Ycf48 attached. Ycf48, a 7-bladed beta propeller, binds to the amino-acid residues of D1 that ultimately ligate the water-oxidising Mn4CaO5 cluster, thereby preventing the premature binding of Mn2+ and Ca2+ ions and protecting the site from damage. Interactions with D2 help explain how Ycf48 promotes assembly of the D1/D2 complex. Overall, our work provides valuable insights into the early stages of PSII assembly and the structural changes that create the binding site for the Mn4CaO5 cluster.
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Journal articleChee MSJ, Serrano E, Chiang YN, et al., 2023,
Dual pathogenicity island transfer by piggybacking lateral transduction
, Cell, Vol: 186, Pages: 3414-3426.e16, ISSN: 0092-8674Lateral transduction (LT) is the process by which temperate phages mobilize large sections of bacterial genomes. Despite its importance, LT has only been observed during prophage induction. Here, we report that superantigen-carrying staphylococcal pathogenicity islands (SaPIs) employ a related but more versatile and complex mechanism of gene transfer to drive chromosomal hypermobility while self-transferring with additional virulence genes from the host. We found that after phage infection or prophage induction, activated SaPIs form concatamers in the bacterial chromosome by switching between parallel genomic tracks in replication bubbles. This dynamic life cycle enables SaPIbov1 to piggyback its LT of staphylococcal pathogenicity island vSaα, which encodes an array of genes involved in host-pathogen interactions, allowing both islands to be mobilized intact and transferred in a single infective particle. Our findings highlight previously unknown roles of pathogenicity islands in bacterial virulence and show that their evolutionary impact extends beyond the genes they carry.
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Journal articleZeden MS, Schuster CF, Gründling A, 2023,
Staphylococcus aureus Colony Polymerase Chain Reaction.
, Cold Spring Harb Protoc, Vol: 2023Here, we describe a protocol for a colony polymerase chain reaction (PCR) method for Staphylococcus aureus The methodology involves the preparation of small S. aureus lysates by using the enzyme lysostaphin to degrade the peptidoglycan layer. These lysates are prepared using a small patch of bacteria grown on LB agar plates, and the lysates can subsequently be used for PCR analyses.
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