Mechanism of Ψ-Pro/C-degron recognition by the CRL2 FEM1B ubiquitin ligase
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Abstract
The E3 ligase-degron interaction determines the specificity of the ubiquitin‒proteasome system. We recently discovered that FEM1B, a substrate receptor of Cullin 2-RING ligase (CRL2), recognizes C-degrons containing a C-terminal proline. By solving several cryo-EM structures of CRL2FEM1B bound to different C-degrons, we elucidate the dimeric assembly of the complex. Furthermore, we reveal distinct dimerization states of unmodified and neddylated CRL2FEM1B to uncover the NEDD8-mediated activation mechanism of CRL2FEM1B. Our research also indicates that, FEM1B utilizes a bipartite mechanism to recognize both the C-terminal proline and an upstream aromatic residue within the substrate. These structural findings, complemented by in vitro ubiquitination and in vivo cell-based assays, demonstrate that CRL2FEM1B-mediated polyubiquitination and subsequent protein turnover depend on both FEM1B-degron interactions and the dimerization state of the E3 ligase complex. Overall, this study deepens our molecular understanding of how Cullin-RING E3 ligase substrate selection mediates protein turnover.
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Acknowledgements: We thank Dr. Yong-Xiang Gao and the Cryo-EM Center at the University of Science and Technology of China for technical support with cryo-EM data collection; Prof. Yarden Opatowsky and Julia Guez-Haddad for gel filtration. This work is supported by the National Key R&D Program of China 2022YFA1303100, the National Natural Science Foundation of China (22137007 and 92253301), the Research Funds of Center for Advanced Interdisciplinary Science and Biomedicine of IHM (QYPY20230022), the Ministry of Science and Technology of China (2022YFC2303700 and 2022YFA1302700), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB0490000), the Center for Advanced Interdisciplinary Science and Biomedicine of IHM (QYPY20220019), and the Fundamental Research Funds for the Central Universities (WK9100000032, WK9100000044, and WK9100000027). C.X. is also supported by the Major/Innovative Program of Development Foundation of Hefei Center for Physical Science and Technology (2021HSC-CIP014); I.K. is supported by the European Research Council (ERC-2020-STG 947709), Israel Science Foundation (ISF Grants No. 2380/21 and 3096/21), Alon Fellowship and Applebaum Foundation. R.T.T. is a Sir Henry Wellcome Postdoctoral Fellow (201387/Z/16/Z) and a Pemberton-Trinity Fellow.
Funder: the National Key R&D Program of China 2022YFA1303100
Funder: the Ministry of Science and Technology of China (2022YFC2303700 and 2022YFA1302700)
Funder: the Ministry of Science and Technology of China (2022YFC2303700 and 2022YFA1302700),the Strategic Priority Research Program of the Chinese Academy of Sciences XDB0490000, the Center for Advanced Interdisciplinary Science Biomedicine of IHM QYPY20220019
