Degrons are elements within protein substrates that mediate the interaction with specific degradation machineries to control proteolysis. Recently, a few classes of C-terminal degrons (C-degrons) that are recognized by dedicated cullin-RING ligases (CRLs) have been identified. Specifically, CRL2 using the related substrate adapters FEM1A/B/C was found to recognize C degrons ending with arginine (Arg/C-degron). Here, we uncover the molecular mechanism of Arg/C-degron recognition by solving a subset of structures of FEM1 proteins in complex with Arg/C-degron-bearing substrates. Our structural research, complemented by binding assays and global protein stability (GPS) analyses, demonstrates that FEM1A/C and FEM1B selectively target distinct classes of Arg/C-degrons. Overall, our study not only sheds light on the molecular mechanism underlying Arg/C-degron recognition for precise control of substrate turnover, but also provides valuable information for development of chemical probes for selectively regulating proteostasis.
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We thank the staff from the BL17B/BL18U1/BL19U1/BL19U2/BL01B beamline45 of National Facility for Protein Science in Shanghai at Shanghai Synchrotron Radiation Facility for assistance during data collection, S.J. Elledge for helpful advice and discussion, D. Wasserman for technical assistance and M. Pagano for kindly providing the plasmids containing cDNAs of human FEM1B and FEM1C. This work is supported by the ‘Strategic Priority Research Program’ of the Chinese Academy of Sciences (grant no. XDB19000000) and the National Natural Science Foundation of China (grant nos. 92053107, 31770806). C.X. is also supported by the Major/Innovative Program of the Development Foundation of the Hefei Center for Physical Science and Technology (2018CXFX007) and the ‘Thousand Young Talent program’. I.K. is supported by Alon fellowship for outstanding young researchers.
The authors declare no competing interests.
Peer review information Nature Chemical Biology thanks David Dougan, Hyun Kyu Song and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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a, Sequence alignment of human FEM1C (NP_064562.1), FEM1A (NP_061178.1), and FEM1B (NP_056137.1). The secondary structures of FEM1C and FEM1B are labelled at the top and bottom of the sequences, respectively. In the structure of SIL1-bound FEM1C, R-1 and K-4 binding residues of FEM1C, as well as their counterparts in FEM1B, are labelled in blue and orange, respectively. Their counterparts in FEM1B are also labeled accordingly. b, Sequence alignment of Arg/C-degrons, including those of SIL1, NS11, OR51B2, Clone13, and CDK5R1. The residues at -1 and -4 positions of SIL1 and their counterparts are marked.
Extended Data Fig. 2 The 2|Fo|–|Fc| omit maps of the fused C-degron peptides in the FEM1C structures contoured at 1.0 σ.
a, SIL1 (-7SLLKELR-1); b, NS11 (-6WDKNLR-1); c, OR51BP2 (-5HRFSR-1); d, Clone13 (-6TQGRAR-1); e, SIL1_R/RG (-7LLKELRG-1); f, The 2|Fo|–|Fc| omit map of CDK5R1 (-8RLLLGLDR-1) bound to FEM1B; g, The 2|Fo|–|Fc| omit map of CDK5R1 (-9KRLLLGLDR-1) bound to FEM1C.
a, Overall structure of NS11-bound FEM1C; b, Overall structure of OR51BP2-bound FEM1C; c, Overall structure of Clone13-bound FEM1C; d, Overall structure of SIL1_R/RG-bound FEM1C; e, Overall structure of CDK5R1-bound FEM1C. The ankyrin repeats and TPR repeats of FEM1C are shown in blue and red cartoon representations, respectively, and the C-degron peptides are shown in yellow cartoon.
a, FEM1C and NS11; b, FEM1C and OR51B2;. c, FEM1C and Clone13; d, FEM1C and SIL1_R/RG; e, FEM1B and CDK5R1; f, FEM1C and CDK5R1. Intermolecular hydrogen bonds and hydrophobic interactions are indicated by black dashes and black arrows, respectively.
a, The electrostatic surface of the FEM1C ankyrin repeats (1-240) bound with OR51B2, with the peptides shown in yellow sticks and labelled. b, Recognition of −4RFSR−1 (R-X-X-R) by FEM1C. The peptide residues and FEM1C residues involved in the intermolecular interactions are labelled, and shown in yellow and blue sticks, respectively. c, The electrostatic surface of the FEM1C ankyrin repeats (1-240) bound with the Clone13 −6TQGRAR−1, d, Recognition of −4GRAR−1 (R-X-R) by FEM1C. e, The electrostatic surface of the FEM1C ankyrin repeats (1-240) bound with the peptide −7LLKELRG−1. f, Recognition of −5KELRG−1 (K-X-X-RG) by FEM1C. g, The electrostatic surface of the FEM1C ankyrin repeats (1-240) bound with CDK5R1. (h) Recognition of −9KRLLLGLDR−1 by FEM1C. (c), (e), and (g) are shown in a manner similar to that shown in Extended Data Fig. 5a, while (d), (f), and (h) are shown in a way similar to that shown in Extended Data Fig. 5b.
Comparison of the binding of K-X-X-R binding mode (SIL1) with that of a, R-F-S-R binding mode (OR51B2), b, G-R-A-R (Clone13), and c, K-X-X-RG (SIL1_R/RG). In (a)–(c), the SIL1 peptide is shown in yellow ribbon, with key reisdues shown in sticks. The SIL1 interaction residues of FEM1C are shown in blue sticks. The peptides of OR51B2, Clone13, and SIL1_R/RG are shown in gray ribbon, with key residues shown in sticks. The FEM1C residues that interact with OR51B2, Clone13, or SIL1_R/RG are shown in grey sticks. Different conformations of key residues are highlighted by red circles.
a, b, ITC binding curves of CDK5R1 binding to FEM1B1-356 (a) and FEM1C1-390 (b). c, d, ITC binding curves of FEM1C1-390 triple mutant H155N/N183A/D188F titrated with SIL1452-461 (c) and CDK5R1298-307 (d).
Extended Data Fig. 9 Comparison of the Arg/C-degron recognition by FEM1C with that of Gly/C-degron by KLHDC2. The electrostatic surface of FEM1C and KHLDC2 are shown.
a, SIL1 binding surface of FEM1C. The SIL1 peptide is shown in yellow ribbon, with Arg-1 and Lys-4 sown in sticks. FEM1C residues involved in the interaction with Arg-1 and Lys-4 are also shwon in sticks. b, di-glycine binding surface of KHLDC2 (PDB: 6DO3). The di-glycine are shown in yellow sticks, and the KLHDC2 residues involved in binding to di-glycine are also shown in sticks.
Supplementary Tables 1–6 and Figs. 1–4.
Representative ITC binding curves
ChemDraw file for Fig.2c
ChemDraw file for Extended Data Fig. 4a
ChemDraw file for Extended Data Fig. 4b
ChemDraw file for Extended Data Fig. 4c
ChemDraw file for Extended Data Fig. 4d
ChemDraw file for Extended Data Fig. 4e
ChemDraw file for Extended Data Fig. 4f
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Chen, X., Liao, S., Makaros, Y. et al. Molecular basis for arginine C-terminal degron recognition by Cul2FEM1 E3 ligase. Nat Chem Biol 17, 254–262 (2021). https://doi.org/10.1038/s41589-020-00704-3