Biology subjects

Buchanan, C. J.

Publications and source records attributed to Buchanan, C. J..

5 recordsLinked to original sources

bioRxiv2024

Methylthio-alkane reductases use nitrogenase metalloclusters for carbon-sulfur bond cleavage

Methylthio-alkane reductases convert methylated sulfur compounds to methanethiol and small hydrocarbons, a process with important environmental and biotechnological implications. These enzymes are classified as nitrogenase-like enzymes, despite lacking the ability to convert dinitrogen to ammonia, raising fundamental questions about the factors controlling their activity and specificity. Here, we present the first molecular structure of the methylthio-alkane reductase, which reveals large metalloclusters, including the P-cluster and the [Fe8S9C]-cluster, previously only found in nitrogenases. Our findings suggest that distinct metallocluster coordination, surroundings, and substrate channels, determine the activity of these related metalloenzymes. This study provides new insights into nitrogen fixation, sulfur-compound reduction, and hydrocarbon production. We also shed light on the evolutionary history of P-cluster and [Fe8S9C]-cluster-containing reductases emerging prior to nitrogenases.

biochemistry↗

bioRxiv2023

Haptenic adducts of beta-lactam antibiotics elicit antibody responses with narrow clonality and specificity

Many classes of small-molecule drugs form protein adducts in vivo, which may elicit antibodies via a classical hapten-carrier-type response, with implications for both allergy and drug sequestration. Although {beta}-lactam antibiotics are a drug class long associated with these phenomena, the molecular determinants of drug-protein conjugation and consequent drug-specific immune responses remain incomplete. Here, we interrogated factors influencing penicilloyl adduct formation and immunogenicity, and used penicillin G (PenG) to probe the B and T cell determinants of drug-specific IgG responses in mice. We identify through deep clonotyping a dominant murine penicilloyl-specific clonal antibody class encompassing phylogenetically related IGHV1, IGHV5 and IGHV10 subgroup gene segments. Through protein NMR and x-ray structural analysis, we determined that adduct specific antibody clones--the MIL series--predominantly recognise the variable side-chain moiety (which for PenG is phenylacetamide) via a hydrophobic pocket, while secondary H-bond contacts with both thiazolidine and the adducted lysine residue is made. As a result, the cross-reactivity against other {beta}-lactam antibiotics is limited. These data demonstrate the relationship between the chemistry of protein-reactive drugs such as penicilloyls, and how their predisposition to generating B cell responses can inform the functional implications at the clonal level. Highlights- PenG readily forms immunogenic adducts on lysine sidechains of diverse self- and non-self proteins including complete serum under physiological conditions. - PenG-protein adduction in vitro or in vivo is sufficient to elicit penicillin-specific IgG responses. - Murine B cell clonotypic responses are characterised by near-uniform antibody binding modes of similar immunogenetic origin. - The dominant murine PenG-specific clonotype is dominated by benzene ring recognition and correlates with serological cross-reactivity profiles.

immunology↗

bioRxiv2023

Engineered display of ganglioside-sugars on protein elicits a clonally and structurally constrained B cell response

Ganglioside sugars, as Tumour-Associated Carbohydrate Antigens (TACAs), are long-proposed targets for vaccination and therapeutic antibody production, but their self-like character imparts immunorecessive characteristics that classical vaccination approaches have to date failed to overcome. One prominent TACA, the glycan component of ganglioside GM3 (GM3g), is over-expressed on diverse tumours. To probe the limits of glycan tolerance, we used protein editing methods to display GM3g in systematically varied non-native presentation modes by attachment to carrier protein lysine sidechains using diverse chemical linkers. We report here that such presentation creates glycoconjugates that are strongly immunogenic in mice and elicit robust antigen-specific IgG responses specific to GM3g. Characterisation of this response by antigen-specific B cell cloning and phylogenetic and functional analyses suggests that such display enables the engagement of a highly restricted naive B cell class with a defined germline configuration dominated by members of the IGHV2 subgroup. Strikingly, structural analysis reveals that glycan features appear to be recognised primarily by antibody CDRH1/2, and despite the presence of an antigen-specific Th response and B cell somatic hypermutation, we found no evidence of affinity maturation towards the antigen. Together these findings suggest a reach-through model in which glycans, when displayed in non-self formats of sufficient distance from a conjugate backbone, may engage glycan ready V-region motifs encoded in the germline. Structural constraints define why, despite engaging the trisaccharide, antibodies do not bind natively-presented glycans, such as when linked to lipid GM3. Our findings provide an explanation for the long-standing difficulties in raising antibodies reactive with native TACAs, and provide a possible template for rational vaccine design against this and other TACA antigens. HighlightsO_LIGM3g synthetically coupled via a longer, orthogonal (from backbone) glycoconjugate (LOG) presentation format (thioethyl-lysyl-amidine) display elicits high-titre IgG responses in mice. C_LIO_LIThe germinal centre experience of LOG glycoconjugate-specific B cell responses is directly influenced by the protein backbone. C_LIO_LIStructural characterisation of the antibody response to LOGs reveals highly restricted germline-encoded glycan-engaging motifs that mediate GM3g recognition. C_LIO_LIFailure of antibodies to bind the native trisaccharide highlights barriers to be overcome for the rational design of anti-TACA antibodies. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=172 SRC="FIGDIR/small/543556v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@b2111org.highwire.dtl.DTLVardef@a81b88org.highwire.dtl.DTLVardef@a78c6aorg.highwire.dtl.DTLVardef@1f3b4b9_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2021

MMMVI: Detecting SARS-CoV-2 Variants of Concern in Metagenomic Samples

MotivationSARS-CoV-2 is the causative agent of the COVID-19 pandemic. Variants of Concern (VOCs) and Variants of Interest (VOIs) are lineages that represent a greater risk to public health, and can be differentiated from the wildtype virus based on unique profiles of signature mutations. Metagenomic sequence analysis of wastewater represents an emerging form of surveillance that can capture early signal for these variants in a community prior to detection through public health testing or genomic surveillance activities. However, because multiple viral genomes are likely to be present in a metagenomic sample, additional analytical scrutiny of the sequencing reads beyond variant calling is needed to increase confidence in diagnostic determinations. ResultsWhere multiple signature mutations are present on a given read, they can be used as enhanced biomarkers to confirm the presence of a VOC/VOI in the sample. We have developed MMMVI, a tool to aggregate and report on the likely presence of a VOC/VOI in a set of metagenomic reads based on the detection of reads bearing multiple signature mutations. AvailabilityMMMVI is implemented in Python, and is available under the MIT licence from https://github.com/dorbarker/voc-identify/ Contactdillon.barker@canada.ca

bioinformatics↗

bioRxiv2021

Cryptic SARS-CoV2-spike-with-sugar interactions revealed by 'universal' saturation transfer analysis

Many host pathogen interactions such as human viruses (including non-SARS-coronaviruses) rely on attachment to host cell-surface glycans. There are conflicting reports about whether the Spike protein of SARS-CoV-2 binds to sialic acid commonly found on host cell-surface N-linked glycans. In the absence of a biochemical assay, the ability to analyze the binding of glycans to heavily- modified proteins and resolve this issue is limited. Classical Saturation Transfer Difference (STD) NMR can be confounded by overlapping sugar resonances that compound with known experimental constraints. Here we present universal saturation transfer analysis (uSTA), an NMR method that builds on existing approaches to provide a general and automated workflow for studying protein-ligand interactions. uSTA reveals that B-origin-lineage-SARS-CoV-2 spike trimer binds sialoside sugars in an end on manner and modelling guided by uSTA localises binding to the spike N-terminal domain (NTD). The sialylated-polylactosamine motif is found on tetraantennary human N-linked-glycoproteins in deeper lung and may have played a role in zoonosis. Provocatively, sialic acid binding is abolished by mutations in some subsequent SARS- CoV-2 variants-of-concern. A very high resolution cryo-EM structure confirms the NTD location and end on mode; it rationalises the effect of NTD mutations and the structure-activity relationship of sialic acid analogues. uSTA is demonstrated to be a robust, rapid and quantitative tool for analysis of binding, even in the most demanding systems. Extended AbstractThe surface proteins found on both pathogens and host cells mediate entry (and exit) and influence disease progression and transmission. Both types can bear host-generated post- translational modifications such as glycosylation that are essential for function but can confound biophysical methods used for dissecting key interactions. Several human viruses (including non- SARS-coronaviruses) attach to host cell-surface N-linked glycans that include forms of sialic acid (sialosides). There remains, however, conflicting evidence as to if or how SARS-associated coronaviruses might use such a mechanism. Here, we demonstrate quantitative extension of saturation transfer protein NMR methods to a complete mathematical model of the magnetization transfer caused by interactions between protein and ligand. The method couples objective resonance-identification via a deconvolution algorithm with Bloch-McConnell analysis to enable a structural, kinetic and thermodynamic analysis of ligand binding beyond previously-perceived limits of exchange rates, concentration or system. Using an automated and openly available workflow this universal saturation transfer analysis (uSTA) can be readily-applied in a range of even heavily-modified systems in a general manner to now obtain quantitative binding interaction parameters (KD, kEx). uSTA proved critical in mapping direct interactions between natural sialoside sugar ligands and relevant virus-surface attachment glycoproteins - SARS-CoV-2-spike and influenza-H1N1-haemagglutinin variants - by quantitating ligand signal in spectral regions otherwise occluded by resonances from mobile protein glycans (that also include sialosides). In B- origin-lineage-SARS-CoV-2 spike trimer end on-binding to sialoside sugars was revealed contrasting with extended surface-binding for heparin sugar ligands; uSTA-derived constraints used in structural modelling suggested sialoside-glycan binding sites in a beta-sheet-rich region of spike N-terminal domain (NTD). Consistent with this, uSTA-glycan binding was minimally- perturbed by antibodies that neutralize the ACE2-binding domain (RBD) but strongly disrupted in spike from the B1.1.7/alpha and B1.351/beta variants-of-concern, which possess hotspot mutations in the NTD. Sialoside binding in B-origin-lineage-NTD was unequivocally pinpointed by cryo-EM to a site that is created from residues that are notably deleted in variants (e.g. H69,V70,Y145 in alpha). An analysis of beneficial genetic variances in cohorts of patients from early 2020 suggests a model in which this site in the NTD of B-origin-lineage-SARS-CoV-2 (but not in alpha/beta-variants) may have exploited a specific sialylated-polylactosamine motif found on tetraantennary human N-linked-glycoproteins in deeper lung. Together these confirm a novel binding mode mediated by the unusual NTD of SARS-CoV-2 and suggest how it may drive virulence and/or zoonosis via modulation of glycan attachment. Since cell-surface glycans are widely relevant to biology and pathology, uSTA can now provide ready, quantitative, widespread analysis of complex, host-derived and post-translationally modified proteins with putative ligands relevant to disease even in previously confounding complex systems.

biophysics↗