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The molecular basis of CULLIN E3 ligase regulation by the COP9 signalosome

Periodic Reporting for period 3 - CsnCRL (The molecular basis of CULLIN E3 ligase regulation by the COP9 signalosome)

Reporting period: 2019-01-01 to 2020-06-30

Binding of structurally diverse CRL ligases to CSN

Cullin-RING E3 ubiquitin ligases (CRLs) are a large family of regulatory protein complexes, responsible for ~20% of intracellular protein degradation in humans. CRLs are divided into six families based on their Cullin constituent (either CUL1, CUL2, CUL3, CUL4A/B or CUL5). Each Cullin binds a RING-domain containing protein (RBX1 or RBX2) and a vast repertoire of adaptor/substrate receptor modules, collectively creating more than 240 distinct CRLs. The structures of CRLs and their substrates vary enormously. All ~240 CRL family members are controlled by the COP9 signalosome complex (CSN). CSN removes NEDD8, an activator protein covalently attached to the Cullin C-terminal domain, and thereby inactivates the ligase. Prior to this work it had been unclear what enables a single complex, CSN, to be a master regulator of CRLs. Using structural biology, we set out to define how CSN recognizes structurally diverse CRLs and regulates them in response to a multitude of different substrates.

General biological questions addressed in the ERC grant: Marcomolecular protein machines are comprised of multiple subunits. How master regulatory complexes operate on different protein substrates comprised of different domains, is an important, unresolved question in structural biology. We use the Cullin-RING system and its interaction with CSN to start addressing this question.

Importance of our finding for human health: Protein degradation through the ubiquitin proteasome system is involved in virtually all biological processes. Accordingly, mutation of the ubiquitin ligases and their regulators are frequently found in cancer cells, as well as in other diseases ranging from neurodegeneration to developmental defects. Understanding these diseases on the molecular level requires atomic-level understanding of the proteins involved.

Recently, the Cullin-RING ligase system has emerged as the ubiquitin system of choice to degrade disease-causing proteins in cells through small molecules that reprogram ubiquitin ligase specificity. These small molecular weight compounds bind to the ligase and enable degradation of new protein substrates, so-called neo-substrates that are only targeted in the presence of the drug. Two classes of these small molecules have emerged: (i) small molecular weight glue-type compounds that attach neo-substrates to the surface of the substrate receptor, and (ii) bi-functional molecules that bind the receptor on one end, and through a linker, engage proteins of interest via a binding warhead on the other and thereby recruit them to the ligase for degradation (also referred to as PROTACs, bi-functional degraders etc.).

Overall objectives: The aim of the ERC grant is to understand regulation of Cullin-RING ligases. The focus is on the Cop9 signalosome, a key regulator of all CRLs. We are also interested in understanding how small molecule drugs can reprogram CRLs and how these drugs interplay with CSN and other CRL regulators.
In the course of the ERC grant, we explored the molecular basis of CSN binding across CRLs by combining single particle electron microscopy with functional studies. Despite striking structural differences among CRLs, we find that CSN engages the highly conserved C-terminal domain of the Cullin subunit (CULCTD) and RBX1. CSN-CRL interactions involving the adaptor/substrate receptor modules differ between different CRLs. CSN binding to CRLs is surprisingly plastic and also accommodates dimeric CRLs. Despite this plasticity, the substrate receptors across CRL family members are uniformly positioned and spaced ~15-20 Å away from CSN. We could show that substrates can access CSN-bound CRL substrate receptors, and that bulky substrates, irrespective of their chemical nature and protein sequence, are able to sterically displace CSN. It is thus the size of the bound CRL substrate, not its identity, that protects CRLs against their inactivation by CSN through a steric mechanism. The effective activity of CSN on each of ~240 CRLs is thereby fine-tuned in response to the prevailing concentration of the respective CLR substrates.

Thalidomide and its second-generation derivatives lenalidomide and pomalidomide, together known as immune modulatory drugs (IMiDs), are potent anticancer drugs that are widely used in the treatment of multiple myeloma and 5q-deletion myelodysplastic syndrome (5q-MDS). IMiDs induce degradation of different proteins upon binding to the CRL4CRBN E3 ubiquitin ligase. In multiple myeloma, IMiD efficacy depends on the ability to degrade the Ikaros/Aiolos transcription factors. In 5q-MDS, however, IMiD are effective through the degradation of casein kinase isoform 1 alpha (CK1A1). How IMiDs induce the degradation of proteins when bound to the CRL4CRBN ligase, and how they interplay with CRL regulators, has remained elusive.

We reported the crystal structure of the ternary complex between the substrate receptor CRBN and the DDB1 adaptor of the CRL4CRBN E3 ligase, in the presence of lenalidomide and the CK1A1 neo-substrate at 2.3 Å resolution. The compound together with a surrounding surface patch on CRBN acts as a molecular glue recruiting CK1A1 to the E3 ligase, where it is juxtaposed to the CUL4/Rbx1 moiety of the active E3 ligase. In biophysical assays, we show that lenalidomide is essential for complex formation, and that CK1A1 binding is not detected in the absence of a compound. Ikaros binding to CRBN likewise depends on the presence of IMiDs. We present mutational studies that suggest that CK1A and Ikaros, in the presence of IMiDs, use largely overlapping binding sites on CRBN. We also demonstrate that the drug-bound Ikaros behaves like a biological substrate to CSN and modulates its binding through the steric mechanism described above.

Structural basis of lenalidomide-induced CK1α degradation by the CRL4(CRBN) ubiquitin ligase.
From these studies, we developed synthetic molecules that reprogram the waste disposal machinery of the cell in such a way that cancer-relevant proteins can be recognized and destroyed by the cell itself, which ultimately leads to the death of certain cancer cells.

The mechanism of action of thalidomide is based on the fact that, thanks to cellular enzymes, disease-causing proteins are declared as waste, which leads to their degradation and thus to the death of cancer cells. In our study we investigated the exact mechanism by which thalidomide recognizes and destroys these proteins. We discovered a 'death signal' in these proteins, which is activated by thalidomide and occurs in slightly altered form in many other disease-causing proteins. Many of these proteins are so-called zinc finger transcription factors, i.e. proteins that can switch genes on or off. Transcription factors have important functions during embryonic development but they can also cause cancer and other diseases if, for example, they are hyperactive or show malfunctions. Up to now, there have been no good, uniform therapeutic approaches for this class of proteins. Our research results suggest that novel drugs can be developed on the basis of thalidomide, which mark disease-relevant zinc finger transcription factors for degradation. These finding came directly out of understanding CRL mechanism, and let to clear translational implications.
Although thalidomide has caused one of the greatest medical disasters of modern times, it has become a blockbusters in cancer therapy. And it looks very much like the revolutionary mechanism of action will lead the way for future therapeutics. Our work underscores the current ambitious efforts in the pharmaceutical industry and various biotechnology companies to harness this mechanism of action, also known as targeted protein degradation, for further diseases.

Our work resulted in multiple publications in Nature, Science, Molecular Cell and Nature Molecular and Structural Biology underlining the fundamental importance of the discoveries made.

The ERC has been used to establish cutting-edge cryo-electron-microscopy capabilities in the laboratory. The CSN-CRL work has laid the foundation and work-flow for ongoing cryo-EM studies, and cryo-EM has now become an indispensable technology in the laboratory.
Architecture of CSN-CRL ligase complexes (A), dimeric CRL3-CSN complexes (B), and mechanism of subst