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Structural basis of Cullin-RING ligase regulation by the COP9 signalosome

Periodic Reporting for period 1 - EMCOP9CRL (Structural basis of Cullin-RING ligase regulation by the COP9 signalosome)

Reporting period: 2015-10-12 to 2017-10-11

The components of the Cullin-RING Ligase (CRLs) E3 ubiquitin ligase family play key roles in a wide range of
cellular processes including stress response, signal transduction, apoptosis and cell cycle progression, and
accordingly, defects in their function and/or regulation are prominent in many pathologies including cancer. The
modular CRL architecture is centred upon one of seven different cullin scaffold proteins which associate on one
side with a RING protein that acts as receptor for an E2 ligase and, on the opposite side, with a substrate receptor
(SR) that confers specificity to the complex. The multiplicity of SRs allows the recognition of many different
substrates by the same CRL catalytic core. CRL-mediated ubiquitination modulates the substrate´s biological
activity and in many cases targets them for proteasomal degradation. The COP9 signalosome (CSN) complex plays
a fundamental role in CRL regulation both by forming stable inhibitory complexes with the CRLs where the E2
ligase and substrate binding sites are occluded, and by enzymatically removing Nedd8 (a homologue of ubiquitin)
from the cullin scaffold subunit, in a process termed deneddylation, that leads to inactivation of CRLs. CRL
regulation by CSN is still an incompletely understood topic mostly because of the lack of high resolution
CSN/CRL structures due to the challenge that the crystallization of multi-protein assemblies of such complexity
represents. Fortunately, technological developments in another structural technique, cryoelectron
microscopy, now allow structure determination of relatively small protein complexes (< 500kDa) to near-atomic
resolution. The aim of this project is to use this powerful technique to reveal very high-resolution structures of several
different CSN/CRL holocomplexes and shed light on the mechanistic aspects of their function.
The work reported here only covers activities from 12/10/2015 to 15/03/2016 due to early termination of the award. Work was initiated on
WP1: month 1-6. Cloning of the CRL complexes. A MultiBac construct for wild type CSN and CSNΔCSN5H138A
lacking deneddylating activity as well as a partial construct of the CRL1 (Cul1/Rbx1) was already available in the
Morris Lab. Work therefore focussed on the cloning the remaining CRL complexes (CRL2: VHL/BC/Cul2/Rbx1; CRL3:
Kctd5/Cul3/Rbx1) in MultiBac vectors. This was initiated but not completed during the period of this award.

Work was also started on WP2: month 3-10. Expression, purification and reconstitution of the CSN/CRL holocomplexes. This stage
included the purification of wtCSN and CSNΔCSN5H138A as stocks for downstream applications as well as the expression,
purification and, where appropriate, in vitro neddylation5 of each CRL complex. This work was initiated but not completed during the period of the award.

Other work packages were not due to start until after the date that the award terminated.
The work described above was taken forward by DR Morris and colleagues and a publication describing the results of this work has been submitted for publication and is currently under review.