LAUSR

Dear Editor, Cullin-RING ligases (CRLs) represent the largest superfamily of multi-subunit ubiquitin E3 ligases and regulate a wide range of cellular functions in all eukaryotes. As a structurally distinct sub-family of CRLs, the CRL4 E3 complexes consists of three well-defined basal subunits— a cullin4 (CUL4A or CUL4B) scaffold protein, a RING finger domain catalytic subunit, RBX1, and a large adaptor subunit, DDB1 (DNA damage-binding protein 1). With a unique triple β-propeller (BPA, BPB, and BPC) topology, DDB1 recruits and coordinates a large variety of substrate receptors, known as DCAFs (DDB1 and Cullin4Associated Factors), which in turn recognize specific substrate proteins for ubiquitination (Fig. 1a). CRL4s play an important role in many essential cellular processes, such as transcription, cell cycle progression, DNA damage repair, and chromatin remodeling, and are frequently hijacked by pathogenic viruses. A number of substrates have been identified for CRL4s, which include p21, Cdt1, DDB2, XPC, and histones. Recent studies have further uncovered several neo-substrates of CRL4, which are recruited to DCAF proteins, such as CRBN and DCAF15, by therapeutic and investigational compounds. Major advances in our understanding of CRL4 function have benefited greatly from early structural studies, which started by revealing the unique architecture of its central component, DDB1. Distinct from the adaptor subunits of other CRLs, DDB1 uses one of its β-propeller domains, BPB, to anchor at the N-terminal region of CUL4A. Its two other β-propeller domains, BPA and BPC, form an interwoven doublepropeller fold that is shaped like a half-open clamshell, and provide a binding surface for DCAF substrate receptors (Fig. 1a). Interestingly, the DDB1 triple propeller has a restrained intrinsic flexibility, which allows a wide range of spatial configuration between the BPB domain and the BPA-BPC double-propeller. DDA1 (DDB1 and DET1 associated 1) has emerged as a fourth evolutionarily conserved basal component of the CRL4 core complex. DDA1 was first identified as a subunit of the plant DDD (DDB1–DET1–DDA1) complex, which binds COP10 and together plays a role in repressing photomorphogenesis. Its animal ortholog was also found in mammalian CRL4s and the DDD-E2 complex (DDB1–DET1–DDA1–UbE2E-E2). While it is well established that aberrant expression of CUL4 is observed in multiple tumors, an increasing number of reports have also suggested a significant contribution of DDA1 as an oncogene. Interestingly, DDA1 has been recently documented to participate in plant hormone abscisic acid signaling and reprogramming of CRL4s by therapeutic compounds for neo-substrate ubiquitination and degradation. Despite this increasing body of evidence implicating DDA1 in CRL4 function and regulation, how it interacts with the other CRL4 subunits and potentially contributes to CRL4 E3 activity remained largely unknown. To better understand the role of DDA1 in CRL4, we first confirmed the direct interaction between DDA1 and DDB1 with purified recombinant proteins and subsequently mapped the region of DDA1 responsible for binding DDB1 (Fig. 1b). Consistent with previous studies, we found that the highly conserved N-terminal 28 amino acids sequence of DDA1 (DDA1-NT) is