Targeted protein degradation is largely performed by the ubiquitin–proteasome pathway, in which substrate proteins are marked by covalently attached ubiquitin chains that mediate recognition by the proteasome. It is currently unclear how the proteasome recognizes its substrates, as the only established ubiquitin receptor intrinsic to the proteasome is Rpn10/S5a (ref. 1), which is not essential for ubiquitin-mediated protein degradation in budding yeast2. In the accompanying manuscript we report that Rpn13 (refs 3–7), a component of the nine-subunit proteasome base, functions as a ubiquitin receptor8, complementing its known role in docking de-ubiquitinating enzyme Uch37/UCHL5 (refs 4–6) to the proteasome. Here we merge crystallography and NMR data to describe the ubiquitin-binding mechanism of Rpn13. We determine the structure of Rpn13 alone and complexed with ubiquitin. The co-complex reveals a novel ubiquitin-binding mode in which loops rather than secondary structural elements are used to capture ubiquitin. Further support for the role of Rpn13 as a proteasomal ubiquitin receptor is demonstrated by its ability to bind ubiquitin and proteasome subunit Rpn2/S1 simultaneously. Finally, we provide a model structure of Rpn13 complexed to diubiquitin, which provides insights into how Rpn13 as a ubiquitin receptor is coupled to substrate deubiquitination by Uch37.
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The help of G. Bourenkov (DESY, BW6, Hamburg, Germany) during synchrotron data collection is gratefully acknowledged. We also thank J. Lary, J. Cole and the National Analytical Ultracentrifugation Facility of the University of Connecticut for performing the sedimentation experiments. NMR data were acquired at the University of Minnesota and the data processed in the Minnesota Supercomputing Institute’s Basic Sciences Computing Laboratory. This work was supported by National Institutes of Health CA097004 (K.W.), GM43601 (D.F.) and GM008700-Chemistry Biology Interface Training Grant (L.R.), Deutsche Forschungsgemeinschaft (DI 931/3-1) and the Cluster of Excellence ‘Macromolecular Complexes’ of the Goethe University Frankfurt (EXC115) to I.D., and Deutsche Forschungsgemeinschaft SFB740/TP B4 (M.G.).
Author Contributions The crystal structure of mRpn13 was solved by P.S. and M.G. (Fig. 1); the complexed structure of mRpn13–ubiquitin (Figs 2a, b and 4), the model structure of mRpn13–diubiquitin (Fig. 3), and NMR studies (Fig. 2d) were conducted by X.C., L.R., N.Z. and K.J.W.; and identification of the interactions, minimal binding domains, protein purification and western blot analysis (Fig. 2c) were performed by K.H. Plasmids were created by K.H. and S.E. I.D., M.G., D.F. and K.J.W. all contributed to design of the experiments and writing the manuscript.
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Schreiner, P., Chen, X., Husnjak, K. et al. Ubiquitin docking at the proteasome through a novel pleckstrin-homology domain interaction. Nature 453, 548–552 (2008). https://doi.org/10.1038/nature06924
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