Conformational Ensembles from Experimental Data and Computer Simulations

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Conformational Ensembles from Experimental Data
and Computer Simulations Poster Abstracts
91
56-POS Board 16
Conformational Dynamics of the Cop9 Signalosome-Cullin-2 RING E3 Ligase
Supercomplex Probed by Hybrid Mass Spectrometry
Andy M. Lau1, Sarah V. Faull2, Chloe Martens 1, Zainab Ahdash1, Edward P. Morris2, Argyris
Politis 1.
2Institute of Cancer Research, London, United Kingdom.1King's College London, London,
United Kingdom,
The Cullin-2 (CUL2) scaffold, along with RING-box binding protein 1 (RBX1) forms the core
of a Cullin-2-RING Ligase (CRL2) complex. Further association with the VHL-EloB/C (VBC)
adaptor complex, the CRL2VBC is responsible for cellular modulation of oxygen-dependent
processes through regulation of the HIF-1α transcription factor. The activity of CRL2VBC is
regulated by the conjugation of the ubiquitin-like protein, NEDD8 (N8), through a process
known as neddylation. Deactivation is achieved through complexing with the hetero-octameric
Cop9 Signalosome (CSN) assembly which removes N8 from the CUL2 scaffold. Despite the
importance of CSN-mediated CRL deactivation in regulating critical cellular processes, the
precise and ubiquitous deactivation mechanism of CSN remains elusive to traditional structural
biology techniques. Using a hybrid mass spectrometry (MS)-based strategy employing native,
ion-mobility, chemical cross-linking and hydrogen deuterium exchange (HDX)-MS, combined
with molecular dynamics simulations and integrative modelling with cryo-EM mapping, we have
documented the highly dynamic conformational responses of CSN upon stimulation by both
neddylated and deneddylated CRL2VBC. Native MS has confirmed the formation of the CSN-
CRL2VBC supercomplex, while a mutation in the CSN5 subunit has allowed the generation of the
CSN-CRL2 VBC~N8 deneddylation intermediate. Integrative modelling of subunit connectivities
via chemical cross-linking MS and electron density maps via cryo-EM have identified discrete
architectures of CSN-CRL2VBC and CSN-CRL2VBC~N8. Using differential HDX-MS and
molecular dynamics simulations, we have further characterised the multiple layers of
conformational dynamics imposed by binding of CRL2VBC and CRL2VBC~N8. Our results show
that neddylation of CRL2VBC directly influences the mode of binding to CSN, while the
deneddylation mechanism of CSN5 is indifferent to the neddylation status.