Biology subjects

Maslen, S. L.

Publications and source records attributed to Maslen, S. L..

10 recordsLinked to original sources

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↗

bioRxiv2026

Molecular determinants of cotranslational chaperone recruitment to nascent p53

Protein folding is fast relative to mRNA translation and nascent polypeptides begin to fold during their synthesis on the ribosome. The resulting cotranslational folding intermediates are accessible to diverse molecular chaperones that recognise incompletely folded client proteins. Here, we sought to understand how specific chaperone:client complexes are established during protein synthesis, using the chaperone-dependent tumor suppressor protein p53 as a model. By capturing interactomes at different points during p53 synthesis, we show that Hsp70, Hsp90 and TRiC bind competitively to nascent p53. While Hsp70 and Hsp90 bind promiscuously, TRiC discriminates between subtly different partially-folded states. TRiC recruitment is dictated by local cotranslational folding and exposure of specific sequence motifs, and this is tuned by cancer-associated mutations in the p53 DNA-binding domain. Nascent chain interactions with the ribosome surface disfavor TRiC binding, demonstrating that the ribosome imposes unique constraints on chaperone recruitment during protein synthesis. Our results establish molecular principles underlying chaperone prioritization during protein biogenesis.

biochemistry↗

bioRxiv2024

The human ribosome modulates multidomain protein biogenesis by delaying cotranslational domain docking

Proteins with multiple domains are intrinsically prone to misfold, yet fold efficiently during their synthesis on the ribosome. This is especially important in eukaryotes, where multidomain proteins predominate. Here, we sought to understand how multidomain protein folding is modulated by the eukaryotic ribosome. We used cryo-electron microscopy and hydrogen/deuterium exchange-mass spectrometry to characterise the structure and dynamics of partially-synthesised intermediates of a model multidomain protein. We find that nascent subdomains fold progressively during synthesis on the human ribosome, templated by interactions across domain interfaces. The conformational ensemble of the nascent chain is tuned by its unstructured C-terminal segments, which keep interfaces between folded domains in dynamic equilibrium until translation termination. This contrasts with the bacterial ribosome, on which domain interfaces form early and remain stable during synthesis. Delayed domain docking may avoid interdomain misfolding to promote the maturation of multidomain proteins in eukaryotes.

biochemistry↗

bioRxiv2024

GroEL/ES chaperonin unfolds then encapsulates a nascent protein on the ribosome

The bacterial chaperonin GroEL/ES promotes protein folding post-translation by transiently encapsulating substrate proteins within a central chamber. GroEL also binds translating ribosomes in vivo, suggesting an additional role in cotranslational folding. Here, we used biochemical reconstitution, structural proteomics and electron microscopy to study the mechanism by which GroEL/ES engages nascent polypeptides. We show that GroEL binds nascent chains on the inside of its cavity via the apical domains and disordered C-terminal tails, resulting in local structural destabilization of the substrate. Ribosome-tethered nascent proteins are partially encapsulated upon GroES binding to GroEL, and refold in the chaperonin cavity. Reconstitution of chaperone competition at the ribosome shows that both Trigger factor and GroEL can be accommodated on long nascent chains, but GroEL and DnaK are mutually antagonistic. Our findings extend the role of GroEL/ES in de novo protein folding, and reveal an unexpected plasticity of the chaperonin mechanism that allows cotranslational substrate encapsulation.

biochemistry↗

bioRxiv2024

Mechanism of chaperone coordination during cotranslational protein folding in bacteria

Protein folding is assisted by molecular chaperones that bind nascent polypeptides during mRNA translation. Several structurally-distinct classes of chaperone promote de novo folding, suggesting that their activities are coordinated at the ribosome. We used biochemical reconstitution and structural proteomics to explore the molecular basis for cotranslational chaperone action in bacteria. We found that chaperone binding is disfavoured close to the ribosome, allowing folding to precede chaperone recruitment. Trigger factor subsequently recognises compact folding intermediates exposing extensive non-native surface and dictates DnaJ access to nascent chains. DnaJ uses a large surface to bind structurally diverse intermediates, and recruits DnaK to sequence-diverse solvent-accessible sites. Neither Trigger factor, DnaJ nor DnaK destabilize cotranslational folding intermediates. Instead, the chaperones collaborate to create a protected space for protein maturation that extends well beyond the ribosome exit tunnel. Our findings show how the chaperone network selects and modulates cotranslational folding intermediates.

biochemistry↗

bioRxiv2023

The V-ATPase/ATG16L1 axis is controlled by the V1H subunit

Defects in organellar acidification indicate compromised or infected compartments. Recruitment of the autophagy-related ATG16L1 complex to pathologically de-acidified compartments targets ubiquitin-like ATG8 molecules to perturbed membranes. How this process is coupled to pH gradient disruption is unclear. Here, we reveal a direct role for the V1H subunit of the V-ATPase proton pump in recruiting ATG16L1. The interaction between V1H and ATG16L1 occurs within assembled V-ATPases, but not dissociated V1 complexes. This selectivity allows recruitment to be coupled to changes in V-ATPase assembly that follow pH dissipation. Cells lacking V1H undergo canonical macroautophagy but are unable to recruit ATG16L1 in response to influenza infection or ionophore drugs. We identify a loop within V1H that mediates ATG16L1 binding, which is absent in a neuronal isoform of V1H. Thus, V1H controls ATG16L1 recruitment in response to proton gradient dissipation, suggesting that the V-ATPase acts autonomously as a cell-intrinsic damage sensor.

molecular biology↗

bioRxiv2022

Trim-Away degrades lysine-less substrates without requiring ligase autoubiquitination

TRIM proteins are the largest family of E3 ligases in mammals. They include the intracellular antibody receptor TRIM21, which is responsible for mediating targeted protein degradation during Trim-Away. Despite their importance, the ubiquitination mechanism of TRIM ligases has remained elusive. Here we show that while TRIM21 activation results in ubiquitination of both ligase and substrate, autoubiquitination is regulatory and not required for substrate degradation. Substrate binding stimulates N-terminal RING autoubiquitination by the E2 Ube2W, but when inhibited by N-terminal acetylation this prevents neither substrate ubiquitination nor degradation and has no impact on TRIM21 antiviral activity. Instead, uncoupling ligase and substrate degradation prevents ligase recycling and extends functional persistence in cells. Substrate ubiquitination mediates degradation but Trim-Away efficiently degrades lysine-less substrates, suggesting a non-canonical ubiquitination mechanism explains its broad substrate specificity.

molecular biology↗

bioRxiv2022

Juxtaposition of Bub1 and Cdc20 on phosphorylated Mad1 during catalytic mitotic checkpoint complex assembly

In response to improper kinetochore-microtubule attachments in mitosis, the spindle assembly checkpoint (SAC) assembles the mitotic checkpoint complex (MCC) to inhibit the anaphase- promoting complex/cyclosome, thereby delaying entry into anaphase. The MCC is a complex of Mad2, Cdc20 and BubR1:Bub3. Its assembly is catalysed by unattached kinetochores on a catalytic Mad1:Mad2 platform. Mad1-bound closed-Mad2 (C-Mad2) recruits open-Mad2 (O- Mad2) through self-dimerization. This interaction, combined with Mps1 kinase-mediated phosphorylation of Bub1 and Mad1, accelerates MCC assembly, in a process that requires conversion of O-Mad2 to C-Mad2 and concomitant binding of Cdc20. How Mad1 phosphorylation catalyses MCC assembly is poorly understood. In this study we characterized Mps1 phosphorylation of Mad1 and obtained structural insights into a phosphorylation-specific Mad1:Cdc20 interaction. This interaction, together with the Mps1-phosphorylation dependent association of Bub1 and Mad1, generates a tripartite assembly of Bub1 and Cdc20 onto the C- terminal domain of Mad1 (Mad1CTD). We additionally identified a folded state of the Mad1:Mad2 complex that suggests how the Cdc20:Mad1CTD interaction brings the Mad2- interacting motif (MIM) of Cdc20 (Cdc20MIM) into close proximity with O-Mad2. We also show that Cdc20MIM is sufficient to catalyse Mad2 conversion and its entrapment by the Mad2 safety- belt. Thus, Mps1-dependent formation of the MCC-assembly scaffold functions to position and orient Cdc20MIM near O-Mad2, thereby catalysing formation of C-Mad2:Cdc20.

biochemistry↗

bioRxiv2020

Sequences in the cytoplasmic tail of SARS-CoV-2 spike facilitate syncytia formation

The Spike (S) protein of SARS-CoV-2 binds ACE2 to direct fusion with host cells. S comprises a large external domain, a transmembrane domain (TMD) and a short cytoplasmic tail. Understanding the intracellular trafficking of S is relevant to SARS-CoV-2 infection, and to vaccines expressing full-length S from mRNA or adenovirus vectors. We have applied proteomics to identify cellular factors that interact with the cytoplasmic tail of S. We confirmed interactions with the COPI and COPII vesicle coats, ERM family actin regulators, and the WIPI3 autophagy component. The COPII binding-site promotes exit from the endoplasmic reticulum (ER), and although COPI-binding should retain S in the early Golgi where viral budding occurs, there is a suboptimal histidine residue in the recognition motif. As a result, S leaks to the surface where it accumulates and can direct the formation of multinucleate syncytia. Thus, the trafficking signals in the tail of S indicate that syncytia play a role in the SARS-CoV-2 lifecycle.

cell biology↗

bioRxiv2019

DEK influences the trade-off between growth and arrest via H2A.Z-nucleosomes in Arabidopsis

The decision of whether to grow and proliferate or to restrict growth and develop resilience to stress is a key biological trade-off. In plants, constitutive growth results in increased sensitivity to environmental stress1,2. The underlying mechanisms controlling this decision are however not well understood. We used temperature as a cue to discover regulators of this process in plants, as it both enhances growth and development rates within a specific range and is also a stress at extremes. We found that the conserved chromatin-associated protein DEK plays a central role in balancing the response between growth and arrest in Arabidopsis, and it does this via H2A.Z-nucleosomes. DEK target genes show two distinct categories of chromatin architecture based on the distribution of H2A.Z in +1 nucleosome and gene body, and these predict induction or repression by DEK. We show that these chromatin signatures of DEK target genes are conserved in human cells, suggesting that DEK may act through an evolutionarily conserved mechanism to control the balance between growth and arrest in plants and animals.

plant biology↗