Biology subjects

Batchelor, M.

Publications and source records attributed to Batchelor, M..

8 recordsLinked to original sources

bioRxiv2026

Molecular mechanism of Aurora-A binding and activation by hnRNPK

Aurora-A kinase (AurA) is a serine/threonine kinase regulated by protein binding partners such as TPX2, which interacts with the Y-pocket of the kinase domain. Although the canonical functions of AurA are mitotic, where it phosphorylates spindle-associated proteins such as TACC3, there is evidence for non-mitotic, chromatin-associated roles. Heterogenous nuclear ribonucleoprotein K (hnRNPK) is a transcriptional regulator and an AurA substrate, how AurA is activated in this pathway is unknown. Their interaction contributes to cancer-associated nuclear accumulation of AurA and has been implicated in driving oncogenic transcription of MYC. Here, we show that hnRNPK promotes AurA autophosphorylation and stimulates AurA activity. The interface was mapped to residues 330-380, an intrinsically disordered region of hnRNPK, and the AurA Y-pocket, using NMR spectroscopy and competitive-binding experiments with mitotic and synthetic AurA-binding partners. AlphaFold3 modelling of the complex predicted Phe339 and Trp345 at the kinase Y-pocket, and site-specific mutagenesis confirmed these as key residues for AurA binding and activation. Our work provides a mechanistic basis for hnRNPK-dependent activation of AurA and efficient phosphorylation of hnRNPK Ser379. Defining the AurA/hnRNPK interface highlights the Y-pocket as a target site for modulation of nuclear AurA, a potentially cancer-specific role of the kinase.

biochemistry↗

bioRxiv2025

Bora Bridges Aurora-A Activation and Substrate Recognition of PLK1

Cell cycle transitions are orchestrated by regulated protein kinase activities. The activation of PLK1 in late G2 is a decisive step for cells to enter mitosis, requiring phosphorylation of its activation loop by Aurora-A, facilitated by the intrinsically disordered protein Bora. Despite its biological importance, the structural basis of this mechanism has remained unresolved. Here, we present models of the Aurora-A/Bora complex and the ternary Aurora-A/Bora/PLK1 complex, validated with site-specific mutagenesis, biochemical assays and NMR spectroscopy. Bora wraps around the N-lobe of Aurora-A, occupying the pockets used by its other activators. A CDK1 phosphorylation site on Bora (Ser112) mimics the structural role of Aurora-A activation loop phosphorylation within a TPX2-like binding motif. In the ternary complex, Bora bridges the two kinases, orienting the activation loop of PLK1 towards the active site of Aurora-A. Bora residues 56-66 form a critical interface with a conserved pocket on the PLK1 C-helix that is analogous to the TPX2-binding Y-pocket of Aurora-A. Aurora-A phosphorylation of Bora Ser59 creates an additional interaction that increases the efficiency of PLK1 phosphorylation. These findings deepen our understanding of how Aurora-A activity is fine-tuned by its disordered binding partners and establish a mechanistic framework for its Bora-dependent activation of PLK1.

molecular biology↗

bioRxiv2024

Mechanism of Interaction Between the Transactivation Domain of N-MYC and the DNA-Binding Surface of TFIIIC5

N-myc is a member of the myc family of transcription factors, which are powerful drivers of cellular growth and consequently, important oncoproteins. N-myc interacts with many factors and complexes to affect transcription. One such complex is the RNA Polymerase III assembly factor, TFIIIC, a six-member complex that is essential for the transcription of small, structured RNA. TFIIIC and N-myc mutually restrict each others chromatin association, and their complex contributes to quality control in mRNA transcription. We previously demonstrated that the largely intrinsically disordered transactivation domain of N-myc interacts directly with a sub-complex of TFIIIC, {tau}A. Structural studies by others show that DNA binding of {tau}A is largely mediated by TFIIIC3, which suggests that TFIIIC5 is at most a secondary binding site for DNA. Here we identified the DNA binding domain of TFIIIC5 as a key binding site for N-myc. We used an integrated approach combining NMR, HDX mass spectrometry, pull-downs and biophysical assays to elucidate the molecular basis of the interaction. Two sequences in the transactivation domain of N-myc bind to the DNA binding interface of TFIIIC5. AlphaFold modelling predicts a high-confidence binding mode for the higher affinity N-myc motif that overlaps with the predicted intramolecular binding site of the C-terminal acidic plug of TFIIIC5, removal of which enhances the binding of N-myc. The same two motifs in N-myc also interact with Aurora-A kinase, which competes with N-myc for TFIIIC binding during S-phase. This model elucidates how the N-myc:TFIIIC5 interaction competes with other interactions, providing a basis for their mutual censoring function and regulation. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=125 SRC="FIGDIR/small/619198v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1e4a76aorg.highwire.dtl.DTLVardef@ab349corg.highwire.dtl.DTLVardef@af85dorg.highwire.dtl.DTLVardef@37dd2f_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

Exploring the dynamics and interactions of the N-myc transactivation domain through solution NMR

The myc family of proteins (c-, N- and L-myc) are transcription factors (TFs) responsible for maintaining the proliferative program in cells. They consist of a C-terminal domain that mediates heterodimerisation with Max and DNA binding, and an N-terminal disordered region culminating in the transactivation domain (TAD). The TAD participates in many protein-protein interactions, notably with kinases that promote stability (Aurora-A) or degradation (ERK1, GSK3) via the ubiquitin-proteasome system. Structural characterization of the TAD of N-myc, is very limited, with the exception of a crystal structure of Aurora-A bound to a helical region of N-myc. We probed the structure, dynamics and interactions of N-myc TAD using nuclear magnetic resonance (NMR) spectroscopy following its complete backbone assignment enabled by a truncation approach. Chemical shift analysis revealed that N-myc has two regions with clear helical propensity: one region within Trp77-Glu86 and the second between Ala122-Glu132. These regions also have more restricted ps-ns motions than the rest of the TAD, and, along with another known interaction site (myc box I), have comparatively high transverse (R2) 15N relaxation rates, indicative of slower timescale dynamics and/or chemical exchange. Collectively these features suggest differential propensities for structure and interaction, either internal or with binding partners, across the TAD. Solution studies on the interaction between N-myc and Aurora-A revealed a previously uncharacterised binding site. The specificity and kinetics of sequential phosphorylation of N-myc by ERK1 and GSK3 were characterised using NMR and showed no significant structural changes through the rest of the TAD. When doubly phosphorylated on residues Ser62 and Thr58, N-myc formed a robust interaction with the Fbxw7-Skp1 complex. Our study provides foundational insights into N-myc TAD dynamics and a backbone assignment that will underpin future work on the structure, dynamics, interactions and regulatory post-translational modifications of this key oncoprotein.

biophysics↗

bioRxiv2022

Loss of the first β-strand of human prion protein generates an aggregation-competent partially "open" form

Prion diseases, a group of incurable, lethal neurodegenerative disorders of mammals including humans, are caused by prions, assemblies of misfolded host prion protein (PrP). The pathway of PrP misfolding is still unclear, though previous data indicate the presence of a structural core in cellular PrP (PrPC), whose cooperative unfolding presents a substantial energy barrier on the path to prion formation. PrP is a GPI-anchored membrane protein, and a number of studies suggest that membrane interactions play an important role in the conversion of PrPC to its disease-associated form, including a transmembrane form of PrP in which a highly conserved region (residues 110 - 136) spans the ER membrane. Insertion of this region results in the detachment of the PrPC first {beta}-strand from the structural core. The effect of this removal on the structure, stability and self-association of the folded domain of PrPC is determined here through a biophysical characterisation of a truncated form of PrPC lacking this region. Whilst markedly destabilised, NMR chemical shifts show that the truncated protein exhibits tertiary structure characteristic of a fully folded protein and retains its native secondary structure elements, including the second strand of the PrP {beta}-sheet, but with altered conformational flexibility in the {beta}2-2 loop and first -helix. The latter is destabilised relative to the other helical regions of the protein, with markedly increased solvent exposure. This truncated form of PrP fibrilises more readily than the native form of the protein. These data suggest a stepwise mechanism, in which a destabilised "open" form of PrPC may be a key intermediate in the refolding to the fibrillar, pathogenic form of the protein.

biophysics↗

bioRxiv2022

Syntaxin 6 delays prion protein fibril formation and prolongs presence of toxic aggregation intermediates

Prions replicate via the autocatalytic conversion of cellular prion protein (PrPC) into fibrillar assemblies of misfolded PrP. While this process has been extensively studied in vivo and in vitro, non-physiological reaction conditions of fibril formation in vitro have precluded the identification and mechanistic analysis of cellular proteins, which may alter PrP self-assembly and prion replication. Here, we have developed a fibril formation assay for recombinant murine and human PrP (23-231) under near-native conditions (NAA) to study the effect of cellular proteins, which may be risk factors or potential therapeutic targets in prion disease. Genetic screening suggests that variants that increase syntaxin-6 expression in the brain (gene: STX6) are risk factors for sporadic Creutzfeldt-Jakob disease (CJD). Analysis of the protein in NAA revealed counterintuitively that syntaxin-6 is a potent inhibitor of PrP fibril formation. It significantly delayed the lag phase of fibril formation at highly sub-stoichiometric molar ratios. However, when assessing toxicity of different aggregation time points to primary neurons, syntaxin-6 prolonged the presence of neurotoxic PrP species. Electron microscopy and super-resolution fluorescence microscopy revealed that, instead of highly ordered fibrils, in the presence of syntaxin-6 PrP formed less-ordered aggregates containing syntaxin-6. These data strongly suggest that the protein can directly alter the initial phase of PrP self-assembly and, uniquely, can act as an anti-chaperone, which promotes toxic aggregation intermediates by inhibiting fibril formation.

biochemistry↗

bioRxiv2021

α-Helix stabilization by co-operative side chain charge-reinforced interactions to phosphoserine in a basic kinase-substrate motif

How cellular functions are regulated through protein phosphorylation events that promote or inhibit protein-protein interactions (PPIs) is key to understanding regulatory molecular mechanisms. Whilst phosphorylation can orthosterically or allosterically influence protein recognition, phospho-driven changes in the conformation of recognition motifs are less well explored. We recently discovered that clathrin heavy chain recognises phosphorylated TACC3 through a helical motif that, in the unphosphorylated protein, is disordered. However, it was unclear whether and how phosphorylation could stabilize a helix in a broader context. In the current manuscript, we address this challenge using poly-Ala based model peptides and a suite of circular dichroism and nuclear magnetic resonance spectroscopies. We show that phosphorylation of a Ser residue stabilizes the -helix in the context of an Arg(i - 3)pSeri Lys(i + 4) triad through charge-reinforced side chain interactions with positive co-operativity, whilst phosphorylation of Thr induces an opposing response. This is significant as it may represent a general method for control of PPIs by phosphorylation; basic kinase-substrate motifs are common with 55 human protein kinases recognising an Arg at a position -3 from the phosphorylated Ser, whilst the Arg(i - 3)pSeri Lys(i + 4) is a motif found in over 2000 human proteins.

biochemistry↗

bioRxiv2020

Brazilin Removes Toxic alpha-Synuclein and Seeding Competent Assemblies from Parkinson Brain by Altering Conformational Equilibrium

Alpha-synuclein (-syn) fibrils, a major constituent of the neurotoxic Lewy Bodies in Parkinsons disease, form via nucleation dependent polymerization and can replicate by a seeding mechanism. Brazilin, a small molecule derived from red cedarwood trees in Brazil, has been shown to inhibit the fibrillogenesis of amyloid-beta (A{beta}) and -syn, prompting our inquiry in its mechanism of action. Here we test the effects of Brazilin on both seeded and unseeded -syn fibril formation and show that the natural polyphenol inhibits fibrillogenesis of -syn by a unique mechanism that is distinct from other polyphenols and is also distinct from its effect on A{beta}. Brazilin preserves the natively unfolded state of -syn by stabilizing the compact conformation of the -syn monomer over the aggregation-competent extended conformation. Molecular docking of Brazilin shows the molecule to interact both with unfolded -syn monomers and with the cross-{beta} sheet structure of -syn fibrils. Brazilin eliminates seeding competence of -syn assemblies from Parkinsons disease patient brain tissue, and treatment of pre-formed fibril assemblies with Brazilin significantly reduces their toxicity in primary neurons. Our findings suggest that Brazilin has substantial potential as a neuroprotective and therapeutic agent for Parkinsons Disease. Highlights- The natural polyphenol Brazilin binds to monomeric, oligomeric and fibrillar -syn - Brazilin shifts the equilibrium away from aggregation-competent monomer conformations - Brazilin inactivates seeding-competent -syn isolated from Parkinson patients brains - Brazilin detoxifies -syn aggregation intermediates and stabilizes mature amyloid fibrils Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=117 HEIGHT=200 SRC="FIGDIR/small/318220v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@447629org.highwire.dtl.DTLVardef@a9871dorg.highwire.dtl.DTLVardef@1e5a83dorg.highwire.dtl.DTLVardef@8843eb_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗