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Conformational switching of the 26S proteasome enables substrate degradation

Nature Structural & Molecular Biology volume 20, pages 781–788 (2013)Cite this article

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Abstract

The 26S proteasome is the major eukaryotic ATP-dependent protease, responsible for regulating the proteome through degradation of ubiquitin-tagged substrates. Its regulatory particle, containing the heterohexameric AAA+ ATPase motor and the essential deubiquitinase Rpn11, recognizes substrates, removes their ubiquitin chains and translocates them into the associated peptidase after unfolding, but detailed mechanisms remain unknown. Here we present the 26S proteasome structure from Saccharomyces cerevisiae during substrate degradation, showing that the regulatory particle switches from a preengaged to a translocation-competent conformation. This conformation is characterized by a rearranged ATPase ring with uniform subunit interfaces, a widened central channel coaxially aligned with the peptidase and a spiral orientation of pore loops that suggests a rapid progression of ATP-hydrolysis events around the ring. Notably, Rpn11 moves from an occluded position to directly above the central pore, thus facilitating substrate deubiquitination concomitant with translocation.

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Figure 1: Conformational transition of the proteasome from a substrate-free to an actively degrading state.
Figure 2: The subnanometer-resolution structure of the substrate-engaged 26S proteasome.
Figure 3: Rpn11 is coaxially aligned with the ATPase pore in the substrate-engaged state.
Figure 4: Substrate-induced rearrangement of the ATPase subunits creates a widened pore and a continuous central channel throughout the enzyme.
Figure 5: Bimodal stabilization of the preengaged or translocation-competent base conformation by the lid.
Figure 6: The translocation-competent conformation of the base exhibits uniform AAA+ domain interfaces.
Figure 7: Rearrangement of the spiral staircase upon substrate engagement.
Figure 8: Structure-based model for substrate engagement and degradation by the 26S proteasome.

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Acknowledgements

We thank C. Bashore (University of California, Berkeley, Berkeley, California, USA) for providing the construct for the G3P model substrate. We thank E. Nogales for thoughtful discussions and for providing access to her EM facility. Finally, we thank the members of the Martin lab for helpful comments. M.E.M. acknowledges support from the American Cancer Society (grant 121453-PF-11-178-01-TBE), and G.C.L. is supported as a Damon Runyon Cancer Research Foundation Fellow (DRG 2055-10). This research was funded in part by the Searle Scholars Program (A.M.), start-up funds from the University of California Berkeley Molecular and Cell Biology Department (A.M.), the US National Institutes of Health (grant R01-GM094497-01A1 to A.M.), the US National Science Foundation CAREER Program (NSF-MCB-1150288 to A.M.) and the Lawrence Berkeley National Laboratory (G.C.L.).

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  1. Gabriel C Lander

    Present address: Present address: Scripps Research Institute, La Jolla, California, USA.,

Authors and Affiliations

  1. Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA

    Mary E Matyskiela & Andreas Martin

  2. Life Sciences Division, Lawrence Berkeley National Laboratory, University of California, Berkeley, Berkeley, California, USA

    Gabriel C Lander

  3. California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, California, USA

    Andreas Martin

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M.E.M. designed, expressed and purified proteasome constructs and performed biochemical experiments. G.C.L. performed the EM, processing and segmentation analyses. All authors contributed to experimental design, data analyses and manuscript preparation.

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Correspondence to Gabriel C Lander or Andreas Martin.

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Supplementary Video 1

Structural rearrangements in the proteasome regulatory particle that occur upon substrate engagement. (MOV 29215 kb)

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Matyskiela, M., Lander, G. & Martin, A. Conformational switching of the 26S proteasome enables substrate degradation. Nat Struct Mol Biol 20, 781–788 (2013). https://doi.org/10.1038/nsmb.2616

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