Proteome integrity depends on the ubiquitin–proteasome system to degrade unwanted or abnormal proteins. In addition to the N-degrons, C-terminal residues of proteins can also serve as degradation signals (C-degrons) that are recognized by specific cullin-RING ubiquitin ligases (CRLs) for proteasomal degradation. FEM1C is a CRL2 substrate receptor that targets the C-terminal arginine degron (Arg/C-degron), but the molecular mechanism of substrate recognition remains largely elusive. Here, we present crystal structures of FEM1C in complex with Arg/C-degron and show that FEM1C utilizes a semi-open binding pocket to capture the C-terminal arginine and that the extreme C-terminal arginine is the major structural determinant in recognition by FEM1C. Together with biochemical and mutagenesis studies, we provide a framework for understanding molecular recognition of the Arg/C-degron by the FEM family of proteins.
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The atomic coordinates and structure factors of FEM1C and the FEM1C–peptide complex have been deposited in the Protein Data Bank (https://www.rcsb.org/) with the accession codes 6XKC and 7JYA, respectively. Source data are provided with this paper.
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We thank W. Tempel and A. Dong for assistance in data collection and structure determination. This work was supported by the National Natural Science Foundation of China (grants 31900865 (C.D.), 32071193 (C.D.), 81974431 (W.M.), 81874039 (X.Y.) and 81771135 (X.Y.)), an NSERC grant (RGPIN-2016-06300 (J.M.)) and the key project of Tianjin Natural Science Grant (19JCZDJC35600 (X.Y.)).
The authors declare no competing interests.
Peer review information Nature Chemical Biology thanks David Dougan and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Extended Data Fig. 1 Comparison of different C-degrons recognition by FEM1C.
a, GST fusion SIL1 peptide immobilized on GST beads, and the pull-down was performed by incubating purified FEM1C (aa 1-371) in the presence of increasing concentrations of REV peptide. b, Stability comparison of the GFP-fused SIL1 and REV degrons by global protein stability assay (GPS experimental design in Fig. 4a). c, Cross-section view of the Arg/C-degron binding pocket. d, Overlay of REV degron (−3RQR−1) and FEM degron (−4KTER−1) binding pockets. The FEM peptide and its interacting residues of FEM1C are shown as cyan and gray sticks, respectively. Hydrogen bonds between FEM peptide and FEM1C are indicated as blue dash lines. The representation of REV-binding mode is same as Fig. 3d. e,f, The electrostatic potential surfaces of the FEM degron (cyan) and the REV degron (yellow) binding pockets. The K-4 of FEM degron and R-3 of REV degron share the same negatively charged binding pocket.
Extended Data Fig. 2 The effects of C-degron sequence contexts on the binding of FEM proteins.
a, Stability comparison of the GFP-fused SIL1 and its E-3R mutant by global protein stability assay. b, The electrostatic properties of R-3 binding pocket in FEM1C (red, negative; blue, positive). R-3 of REV degron is shown as yellow stick, and its hydrogen-bonding residues in FEM1C are indicated. c, Sequence alignment of FEM1C (aa 182-191), FEM1B (aa 187-196) and FEM1A (aa 183-192). The R-3 interacting residues are colored in red. d, Stability comparison of the GFP-fused REV degron and REV degron capped with a leucine by global protein stability assay. e, f, ITC curve of FEM1C (aa 1-371) binding to the REV degron capped with two serine (e) or three serine residues (f).
Extended Data Fig. 3 Structural comparison of Arg/C-degron and Arg/N-degron recognitions.
a, The electrostatic potential surface of the UBR domain (PDB: 3NIH) bound to an Arg/N-degron plotted at ± 5 kT/e (red, negative; blue, positive). b, The electrostatic potential surface of FEM1C bound to an Arg/C-degron plotted at ± 5 kT/e. c, Interaction of the UBR domain with a Arg/N-degron. The N-terminal arginine is shown as yellow stick and its interacting residues in UBR are shown as salmon sticks. d, Interactions of FEM1C with an Arg/C-degron. The C-terminal arginine is shown as yellow stick and its interacting residues in FEM1C are shown as green sticks.
Supplementary Tables 1–3 and Fig. 1.
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Source Data Fig. 1
Unprocessed gel linked to Fig. 1b.
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Unprocessed gel linked to Fig. 2a.
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Unprocessed western blots linked to Fig. 4b.
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Fluorescence-activated cell sorting data linked to Fig. 4c.
Source Data Extended Data Fig. 1
Unprocessed gel linked to Extended Data Fig. 1a.
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Yan, X., Wang, X., Li, Y. et al. Molecular basis for ubiquitin ligase CRL2FEM1C-mediated recognition of C-degron. Nat Chem Biol 17, 263–271 (2021). https://doi.org/10.1038/s41589-020-00703-4
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