The molecular basis of CRL4 ubiquitin ligase architecture, targeting and regulation

Members of the CUL4-RBX1-DDB1 (CRL4) E3 ubiquitin ligase family regulate multiple cellular processes including development, transcription, and DNA repair. CRL4 type ligases are modularly assembled with specialized substrate receptors (DCAFs), which recruit a specific substrate and thereby confer specificity. Tight regulation of the versatile CRL4 network is crucial to maintain the integrity of important cellular pathways, as deregulation and mutations are often associated with human disease and cancer. A number of regulatory factors for the CRL system have been identified, including the COP9 signalosome (CSN), which has emerged as the major cullin regulator. CSN is a large protease that cleaves the isopeptide bond between a cullin and the ubiquitin-like modifier Nedd8, thereby controlling CRL4 activity. One of the best characterized DCAFs is the DNA damage binding protein 2 (DDB2), which, as part of the CRL4DDB2 ligase, orchestrates the initial steps of nucleotide excision repair (NER). In the first part of my thesis, I applied structural, biochemical and functional methods to elucidate the molecular architecture, targeting, and regulation of this important ligase complex. I demonstrate that CRL4DDB2 is recruited to UV-induced lesions in chromatin and provide the structures of the fully assembled CUL4A/B-RBX1-DDB1-DDB2 ligase complexes bound to damaged DNA substrates. These studies reveal the intrinsic mobility of the ligase arm that creates an ubiquitination zone around the substrate binding site. The distance observed between the E2 binding site and the DNA binding site, together with the mobility of the ligase, preclude direct ligase activation through DNA damage binding. Instead, we found that CSN mediates CRL4DDB2 inhibition in a CSN5 independent fashion and this inhibition is relieved upon the binding of damaged DNA to the DDB2 receptor. We show that the CRL4CSA ligase, involved in transcription coupled repair, shares common architectural features with CRL4DDB2. And that the CSB protein substrate is sufficient to relieve CSN mediated CRL4CSA inhibition. Our data argues in favor of a general mechanism in which CRL4DCAF(WD40) ligases are inhibited by CSN, and in turn, activation of the ligase is induced by substrate binding to, together with CSN displacement from, the ligase. The ubiquitin proteasome system (UPS) controls timely degradation of short-lived proteins, including prominent oncogenes and tumor suppressors such as p53, β-catenin or c-myc. Targeting the UPS has emerged as a promising anti-cancer strategy and the FDA approval of bortezomib as the “first in class” proteasome inhibitor reflects this. However, global inhibition of the proteasome induces dramatic effects on protein homeostasis and high levels of cell toxicity, promoting efforts to target the UPS upstream of the proteasome, particularly through specific CRLs. Despite being known to cause multiple birth defects, thalidomide (contergan) and its more potent derivative lenalidomide (revlimid) are FDA approved and widely used in the treatment of multiple myeloma. While the more recent discovery of the CRL4CRBN ubiquitin ligase as a primary cellular target of thalidomide has been a milestone in understanding thalidomide teratogenicity, a detailed molecular understanding of thalidomide action remains to be elucidated. In the second part of my thesis, I provide the X-ray structure of the DDB1-CRBN heterodimer in complex with the small molecule inhibitors thalidomide, lenalidomide and pomalidomide. The structure provides a molecular rationale for thalidomide action and, for the first time, a structural rationale for specifically targeting a CRL4 E3 ubiquitin ligase.