Biology subjects

Burgess, A. E.

Publications and source records attributed to Burgess, A. E..

4 recordsLinked to original sources

bioRxiv2024

DET1 dynamics underlie co-operative ubiquitination by CRL4DET1 COP1 complexes

Ubiquitin ligases regulate core cellular processes through diverse mechanisms. The ubiquitin ligase COP1 is conserved from plants to humans and is particularly important for targeting developmental transcription factors for ubiquitination. COP1 can function independently, but can also be recruited to Cullin-4 ubiquitin ligase complexes via the DET1 adaptor protein. However, the mechanism of action of complexes containing COP1 and DET1 is not well understood. Here we report the cryo-electron microscopy structure of human DET1, bound to proteins that enable Cullin-4 recruitment (DDB1-DDA1) and an additional ubiquitin ligase enzyme (Ube2e2). We observe that DDA1 stabilises a closed conformation of DET1, binding adjacent to a unique Ube2e2 binding-insert in DET1. Moreover, we demonstrate that closure of DET1 underlies COP1 recruitment, which binds in an antiparallel dimeric state. Disrupting either the Ube2e2-binding insertion of DET1, or distinct recruitment sites on COP1, abolish DET1-COP1 binding and DET1-mediated modulation of COP1 levels. The multifaceted architecture provides an efficient platform for ubiquitination of substrates, or COP1 itself, by Cullin-4DET1 and offers multiple opportunities for physiological regulation.

biochemistry↗

bioRxiv2021

Oncogenic truncations of ASXL1 enhance a motif for BRD4 ET-domain binding

Proper regulation of gene-expression relies on specific protein-protein interactions between a myriad of epigenetic regulators. As such, mutation of genes encoding epigenetic regulators often drive cancer and developmental disorders. Additional sex combs-like protein 1 (ASXL1) is a key example, where mutations frequently drive haematological cancers and can cause developmental disorders. It has been reported that nonsense mutations in ASXL1 promote an interaction with BRD4, another central epigenetic regulator. Here we provide a molecular mechanism for the BRD4-ASXL1 interaction, demonstrating that a motif near to common truncation breakpoints of ASXL1 contains an epitope that binds the ET domain within BRD4. Binding-studies show that this interaction is analogous to common ET-binding modes of BRD4-interactors, and that all three ASX-like protein orthologs (ASXL1-3) contain a functional ET-domain-binding epitope. Crucially, we observe that BRD4-ASXL1 binding is markedly increased in the prevalent ASXL1Y591X truncation that maintains the BRD4-binding epitope, relative to full-length ASXL1 or truncated proteins that delete the epitope. Together, these results show that ASXL1 truncation enhances BRD4 recruitment to transcriptional complexes via its ET domain, which could misdirect regulatory activity of either BRD4 or ASXL1 and may inform potential therapeutic interventions.

biochemistry↗

bioRxiv2019

Mechanism of preferential complex formation by Apoptosis Signal-regulating Kinases

Apoptosis signal-regulating kinases (ASK1-3) are activators of the P38 and JNK MAP kinase pathways. ASK1-3 form oligomeric complexes known as ASK signalosomes that initiate signalling cascades in response to diverse stress stimuli. Here we demonstrate that oligomerization of ASK proteins is driven by previously uncharacterised sterile-alpha motif (SAM) domains that reside at the C-terminus of each ASK protein. SAM domains from ASK1-3 have distinct behaviours: ASK1 forms unstable oligomers, ASK2 is predominantly monomeric, and the ASK3 SAM domain forms a stable oligomer even at low concentration. In contrast to their isolated behaviour, the ASK1 and ASK2 SAM domains preferentially form a stable heterocomplex. The crystal structure of the ASK3 SAM domain, small-angle X-ray scattering, and mutagenesis suggests that ASK3 oligomers and ASK1-ASK2 complexes form discrete quasi-helical rings, via the mid-loop-end-helix interface. Preferential ASK1-ASK2 binding is consistent with mass spectrometry showing that full-length ASK1 forms heterooligomeric complexes incorporating high levels of ASK2. Accordingly, disruption of SAM domain-association impairs ASK activity in the context of electrophilic stress induced by 4-hydroxy-2-nonenal. These findings provide a structural template for how ASK proteins assemble foci to drive inflammatory signalling, and reinforce that strategies targeting ASK kinases should consider the concerted actions of multiple ASK family members.

biochemistry↗

bioRxiv2018

Substrate binding allosterically relieves autoinhibition of the TRIB1 pseudokinase

One Sentence SummarySubstrate binding to Tribbles-homolog 1 (TRIB1) pseudokinase induces allosteric changes that allow formation of a complex with the COP1 ubiquitin ligase.\n\nAbstractThe Tribbles family of pseudokinases recruit substrates to the COP1 ubiquitin ligase for ubiquitination. CCAAT-enhancer binding protein (C/EBP) family transcription factors are crucial Tribbles substrates in adipocyte and myeloid development. Here we show that the TRIB1 pseudokinase can recruit various C/EBP family members, with binding of C/EBP{beta} attenuated by phosphorylation. To explain the mechanism of substrate recruitment, we solved the crystal structure of TRIB1 in complex with C/EBP. TRIB1 undergoes a significant conformational change relative to its substrate-free structure, to bind C/EBP in a pseudo-substrate-like manner. Crucially, substrate binding triggers allosteric changes that link substrate recruitment to COP1 binding, which is consistent with molecular dynamics and biochemical studies. These findings offer a view of pseudokinase regulation with striking parallels to bona fide kinase regulation-- via the activation loop and C-helix--and raise the possibility of small molecules targeting either the activation loop-in, or loop-out, conformations of Tribbles pseudokinases.

biochemistry↗