Structural Basis for Substrate Selectivity of the E3 Ligase COP1

Abstract

Summary COP1 proteins are E3 ubiquitin ligases that regulate phototropism in plants and target transcription factors for degradation in mammals. The substrate-binding region of COP1 resides within a WD40-repeat domain that also binds to Trib proteins, which are adaptors for C/EBPα degradation. We report here structures of the human COP1 WD40 domain in isolation, and complexes of the human and Arabidopsis thaliana COP1 WD40 domains with the binding motif of Trib1. The human and Arabidopsis WD40 domains are seven-bladed β-propellers with an inserted loop on the bottom face of the first blade. The Trib1 peptide binds in an extended conformation to a highly conserved surface on the top face of the β-propeller, indicating a general mode for recognition of peptide motifs by COP1. Together, these studies identify the structural basis and key interactions for motif recognition by COP1, and hint at how Trib1 autoinhibition is overcome to target C/EBPα for degradation.

STK40 Is a Pseudokinase that Binds the E3 Ubiquitin Ligase COP1

“…We have determined that the STK40 C-terminal peptide (339–351) recapitulates binding in a purified system and contributes to binding in cells, and that the COP1 WD40 domain is necessary and sufficient for binding. These results echo our recent findings for the binding of the pseudokinase Trib1 to the WD40 domain of COP1 (Uljon et al, 2016), though the functional significance of the STK40-COP1 interaction is still unknown. Intriguingly, the observation that both STK40 and Tribbles pseudokinases bind to the same site on the COP1 WD40 domain, together with the lack of evidence that either is a target of COP1-mediated degradation, raises the possibility that STK40 and Tribbles may act as adaptors for COP1 substrate selection or as regulators of COP1 ligase activity.…”

“…This motif aligns well to the COP1-binding sequence of Trib1 and contains the signature VP sequence structurally critical for peptide binding (Uljon et al, 2016), but differs from the “canonical” COP1 binding motif because it lacks the acidic residue(s) typically found 2–3 amino acids N-terminal to the VP(D/E) motif. We first tested whether this region of STK40 binds to COP1 in a pull-down assay using a 12 residue peptide fused to GFP, and observed that the isolated peptide recovers the COP1 WD40 domain, but less efficiently than full-length STK40 (Figure 1C, lane 8).…”

“…The STK40 peptide bound to the WD40 domain of COP1 with a K d of 3.1 ± 0.6 µM. This affinity is not as strong as that of an analogous peptide from Trib1 (Uljon et al, 2016), which binds with a K d of 0.5 ± 0.1 µM under these conditions. In competition assays using purified STK40 proteins, the kinase homology domain (KHD) of STK40 does not compete with peptide binding (and appears to increase the polarization change).…”

“…Conversely, a construct of STK40 containing only the central kinase homology domain (KHD) is unable to recover detectable quantities of the COP1 WD40 domain (Figure 1C, Lane 7). We then determined the affinity of the isolated STK40 peptide for the purified COP1 WD40 domain using a previously described fluorescence polarization assay (Uljon et al, 2016). The STK40 peptide bound to the WD40 domain of COP1 with a K d of 3.1 ± 0.6 µM.…”

COP1/DET1/ETS axis regulates ERK transcriptome and sensitivity to MAPK inhibitors

“…We engineered these missense mutations as well as several missense mutations in other cancer types that face the ligand-binding pocket (Table 3) (38). To evaluate whether the point mutations affect the function of COP1, we generated CRISPR/Cas9-mediated COP1 knockout in A375 cells stably expressing the mCherry-nETV1 protein stability sensor (see Supplemental Figure 5D).…”