Biology subjects

Briggs, D. C.

Publications and source records attributed to Briggs, D. C..

8 recordsLinked to original sources

bioRxiv2026

Discovery of a sulfotyrosine-motif in the human TrkB extracellular domain required for agonist activation

The brain-derived neurotrophic factor (BDNF)-tropomyosin receptor kinase B (TrkB) signalling axis is a key effector of synaptic plasticity and neuroprotection. While TrkB activation is a major objective towards preventing dysfunction of the nervous system, it cannot be reached with exogenous BDNF administration given the unfavourable physiochemical properties of BDNF. In addition, BDNF also activates a tumour necrosis factor pathway by binding to the neurotrophin receptor p75. The TrkB agonist ZEB85 provides an alternative route to the selective activation of TrkB. We report here the structural basis for the interaction between human TrkB, and both ZEB85 and BDNF, and reveal that a sulfated tyrosine modification is indispensable for ZEB85 activation of TrkB signalling. Using structure-guided BDNF- and ZEB85-binding deficient TrkB mutants, we assessed their ability to sequester ligands from full-length TrkB in cultured human neurons. We found that the BDNF binding site extends into the extracellular juxtamembrane domain of TrkB but does not require the sulfotyrosine at residue 400 to activate TrkB. Together with biophysical analysis and AlphaFold modelling these results also explain how BDNF can displace ZEB85 from TrkB through an overlapping epitope. Our findings reveal unique features of TrkB, not present in the related neurotrophin receptors TrkA and TrkC, and suggest new directions to explore the role of sulfotyrosine in TrkB signalling and identify new TrkB-specific protein ligands. One Sentence SummaryInvestigation of the mechanism of action of TrkB agonist ZEB85 extends molecular understanding of TrkB activation.

biochemistry↗

bioRxiv2026

A long-chain heparan sulfate capture mechanism directs paracrine GDNF-GFRα1 signalling through RET

The receptor tyrosine kinase RET is activated by GDNF and GFR1 together as a bipartite ligand, driving receptor activation and signalling in developmental and neuroprotective contexts. Evidence from developmental and cell models has suggested that heparan sulfate (HS) functions as a fourth component in RET signalling by binding to GDNF, but the molecular details remain unclear. Here, we present the cryo-EM structure of the heterohexameric RET:GDNF:GFR1 complex with a fully resolved heparin ligand, revealing an unexpected extended HS binding site spanning all three proteins. The architecture of the complex and binding mode of the HS chain in this complex enables the formation of a higher order 4:4:4 assembly bound to a single 30-saccharide HS chain which bridges two intimately bound complexes. This multi-protein interface selectively binds the highly sulfated domains of HS over other GAG classes, and is essential for RET activation in trans with soluble GFR1, but not in cis when GFR1 is membrane-bound. Our data suggest that HS shapes the dynamics of RET signalling at every stage, from ligand diffusion to signalling complex formation. Thus, GDNF-GFRa1 paracrine signalling reveals a surprising dependence for long-chain GAG function in which the glycan engages in both receptor complex assembly and clustering.

biochemistry↗

bioRxiv2024

Capture, mutual inhibition and release mechanism for aPKC-Par6 and its multi-site polarity substrate Lgl

The mutually antagonistic kinase-substrate relationship between the apical aPKC-Par6 heterodimer and the basolateral substrate Lgl is key to the establishment and maintenance of cell polarity across metazoa. Although aPKC-Par6 can phosphorylate Lgl at three serine sites to exclude it from the apical domain, paradoxically, aPKC-Par6 and Lgl can also form a stable kinase-substrate complex whose function remains unclear and with conflicting roles proposed for Par6. We report the structure of human aPKC{iota}-Par6 bound to full-length Llgl1, captured through an aPKC{iota} docking site and a Par6PDZ contact. This soluble tripartite complex traps a phospho-S663 Llgl1 intermediate bridging between aPKC and Par6, impeding phosphorylation progression. Thus, aPKC{iota} is effectively inhibited by Llgl1pS663 whilst Llgl1 is captured by aPKC{iota}-Par6. Mutational disruption of Lgl-aPKC interaction impedes complex assembly and Lgl phosphorylation, whereas disrupting the Lgl-Par6PDZ contact promotes complex dissociation and completion of Lgl phosphorylation cycle. We incorporate these findings into a Par6PDZ-regulated substrate capture-and-release model that we demonstrate requires binding by Cdc42-GTP and the apical partner Crumbs to drive complex disassembly. Our results provide an explanation for the opposing roles of Par6 underpinning the spatial control of aPKC-Par6 activity by Lgl relevant to polarised membrane contexts across multi-cellular organisms.

biochemistry↗

bioRxiv2023

Xylosyltransferase Bump-and-hole Engineering to Chemically Manipulate Proteoglycans in Mammalian Cells

Mammalian cells orchestrate signalling through interaction events on their surfaces. Proteoglycans are an intricate part of these interactions, carrying large glycosaminoglycan polysaccharides that recruit signalling molecules. Despite their importance in development, cancer and neurobiology, a relatively small number of proteoglycans have been identified. In addition to the complexity of glycan extension, biosynthetic redundancy in the first protein glycosylation step by two xylosyltransferase isoenzymes XT1 and XT2 complicates annotation of proteoglycans. Here, we develop a chemical genetic strategy that manipulates the glycan attachment site of cellular proteoglycans. By employing a tactic termed bump- and-hole engineering, we engineer the two isoenzymes XT1 and XT2 to specifically transfer a chemically modified xylose analogue to target proteins. The chemical modification contains a bioorthogonal tag, allowing the ability to visualise and profile target proteins modified by both transferases in mammalian cells. The versatility of our approach allows pinpointing glycosylation sites by tandem mass spectrometry, and exploiting the chemical handle to manufacture proteoglycans with defined GAG chains for cellular applications. Engineered XT enzymes permit a view into proteoglycan biology that is orthogonal to conventional techniques in biochemistry.

biochemistry↗

bioRxiv2022

The impact of AlphaFold on experimental structure solution

AlphaFold2 is a machine-learning based program that predicts a protein structure based on the amino acid sequence. In this article, we report on the current usages of this new tool and give examples from our work in the Coronavirus Structural Task Force. With its unprecedented accuracy, it can be utilized for the design of expression constructs, de novo protein design and the interpretation of Cryo-EM data with an atomic model. However, these methods are limited by their training data and are of limited use to predict conformational variability and fold flexibility; they also lack co-factors, posttranslational modifications and multimeric complexes with oligonucleotides. They also are not always perfect in terms of chemical geometry. Nevertheless, machine learning based fold prediction are a game changer for structural bioinformatics and experimentalists alike, with exciting developments ahead.

bioinformatics↗

bioRxiv2020

A two-site flexible clamp mechanism for RET-GDNF-GFRα1 assembly reveals both conformational adaptation and strict geometric spacing

RET receptor tyrosine kinase plays vital developmental and neuroprotective roles in metazoans. GDNF family ligands (GFLs) when bound to cognate GFR co-receptors recognise and activate RET stimulating its cytoplasmic kinase function. The principles for RET ligand-co-receptor recognition are incompletely understood. Here we report a crystal structure of the cadherin-like module (CLD1-4) from zebrafish RET revealing interdomain flexibility between CLD2-CLD3. Comparison with a cryo-EM structure of a ligand-engaged zebrafish RETECD-GDNF-GFR1 complex indicates conformational changes within a clade-specific CLD3 loop adjacent to co-receptor. Our observations indicate RET is a molecular clamp with a flexible calcium-dependent arm that adapts to different GFR co-receptors, while its rigid arm recognises a GFL dimer to align both membrane-proximal cysteine-rich domains. We also visualise linear arrays of RETECD-GDNF-GFR1 suggesting a conserved contact stabilises higher-order species. Our study reveals ligand-co-receptor recognition by RET involves both receptor plasticity and strict spacing of receptor dimers by GFL ligands. HighlightsO_LICrystal structure of zebrafish RET cadherin-like module reveals conformational flexibility at the calcium-dependent CLD2-CLD3 interface C_LIO_LIComparison of X-ray and cryo-EM structures indicate conformational differences between unliganded and liganded RET involving a clade-specific CLD3 loop C_LIO_LIStrict spatial separation of RETECD C-termini is imposed by each cysteine-rich domain interaction with GFL dimer C_LIO_LIDifferences in co-receptor engagement and higher-order ligand-bound RET complexes indicate potentially divergent signalling mechanisms C_LI

biophysics↗

bioRxiv2020

Metabolic precision labeling enables selective probing of O-linked N-acetylgalactosamine glycosylation

Protein glycosylation events that happen early in the secretory pathway are often dysregulated during tumorigenesis. These events can be probed, in principle, by monosaccharides with bioorthogonal tags that would ideally be specific for distinct glycan subtypes. However, metabolic interconversion into other monosaccharides drastically reduces such specificity in the living cell. Here, we use a structure-based design process to develop the monosaccharide probe GalNAzMe that is specific for cancer-relevant Ser/Thr-N-acetylgalactosamine (O-GalNAc) glycosylation. By virtue of a branched N-acylamide side chain, GalNAzMe is not interconverted by epimerization to the corresponding N-acetylglucosamine analog like conventional GalNAc-based probes. GalNAzMe enters O-GalNAc glycosylation but does not enter other major cell surface glycan types including Asn (N)-linked glycans. We equip cells with the capacity to biosynthesize the nucleotide-sugar donor UDP-GalNAzMe from a caged precursor. Tagged with a bioorthogonal azide group, GalNAzMe serves as an O-glycan specific reporter in superresolution microscopy, chemical glycoproteomics, a genome-wide CRISPR knock-out (KO) screen, and imaging of intestinal organoids. GalNAzMe is a precision tool that allows a detailed view into the biology of a major type of cancer-relevant protein glycosylation. Significance statementA large portion of all secreted and cell surface proteins in humans are modified by Ser/Thr(O)-linked glycosylation with N-acetylgalactosamine (GalNAc). While of fundamental importance in health and disease, O-GalNAc glycosylation is technically challenging to study because of a lack of specific tools to be used in biological assays. Here, we design an O-GalNAc specific reporter molecule termed GalNAzMe to selectively label O-GalNAc glycoproteins in living human cells. GalNAzMe is compatible with a range of experiments in quantitative biology to broaden our understanding of glycosylation. We further demonstrate that labeling is genetically programmable by expression of a mutant glycosyltransferase, allowing application even to experiments with low inherent sensitivity.

synthetic biology↗

bioRxiv2019

Cryo-EM Structures of the XPF-ERCC1 Endonuclease Reveal an Auto-Inhibited Conformation and the Basis for Activation

The structure-specific endonuclease XPF-ERCC1 participates in multiple DNA damage repair pathways including nucleotide excision repair (NER) and inter-strand crosslink repair (ICLR). How XPF-ERCC1 is catalytically activated by DNA junction substrates is not currently understood. We report cryo-electron microscopy structures of both DNA-free and DNA-bound human XPF-ERCC1. DNA-free XPF-ERCC1 adopts an auto-inhibited conformation in which the XPF helical domain masks ERCC1 DNA-binding elements and restricts access to the XPF catalytic site. Binding of a model DNA junction separates the XPF helical and ERCC1 (HhH)2 domains, promoting activation. Using these structural data, we propose a model for a 5-NER incision complex involving XPF-ERCC1-XPA and a DNA junction substrate. Structure-function data suggest xeroderma pigmentosum patient mutations often compromise the structural integrity of XPF-ERCC1. Fanconi anaemia patient mutations often display substantial in-vitro activity but are resistant to activation by ICLR recruitment factor SLX4. Our data provide insights into XPF-ERCC1 architecture and catalytic activation.

molecular biology↗