Abstract
The 26S proteasome is an ATP-dependent dynamic 2.5 MDa protease that regulates numerous essential cellular functions through degradation of ubiquitinated substrates. Here we present a near-atomic-resolution cryo-EM map of the S. cerevisiae 26S proteasome in complex with ADP-AlFx. Our biochemical and structural data reveal that the proteasome-ADP-AlFx is in an activated state, displaying a distinct conformational configuration especially in the AAA-ATPase motor region. Noteworthy, this map demonstrates an asymmetric nucleotide binding pattern with four consecutive AAA-ATPase subunits bound with nucleotide. The remaining two subunits, Rpt2 and Rpt6, with empty or only partially occupied nucleotide pocket exhibit pronounced conformational changes in the AAA-ATPase ring, which may represent a collective result of allosteric cooperativity of all the AAA-ATPase subunits responding to ATP hydrolysis. This collective motion of Rpt2 and Rpt6 results in an elevation of their pore loops, which could play an important role in substrate processing of proteasome. Our data also imply that the nucleotide occupancy pattern could be related to the activation status of the complex. Moreover, the HbYX tail insertion may not be sufficient to maintain the gate opening of 20S core particle. Our results provide new insights into the mechanisms of nucleotide-driven allosteric cooperativity of the complex and of the substrate processing by the proteasome.
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Acknowledgements
We thank Drs Jinqiu Zhou and Zhaocai Zhou (Institute of Biochemistry and Cell Biology, Shanghai) for their generous support. We thank Yingyi Zhang and Mi Cao from the Electron Microscopy facility and Min Xue from Data Base & Computation system at National Centre for Protein Science Shanghai (NCPSS) for their assistance with the EM instrument management and data storage and parallel computing. We are grateful to the NCPSS Protein Expression and Purification facility, Mass Spectrometry facility, and BL19U2 beamline at NCPSS and Shanghai Synchrotron Radiation Facility (SSRF) for instrument support and technical assistance. This work was supported by grants from the CAS Pilot Strategic Science and Technology Projects B (XDB08030201), the National Natural Science Foundation of China (31270771, 31670754), the National Basic Research Program of China (2013CB910401), the STS program of the CAS (KFJ-EW-STS-098), and the CAS-Shanghai Science Research Center (CAS-SSRC-YH-2015-01).
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Supplementary information
Supplementary information, Figure S1
Biochemical analyses of the yeast proteasome. (PDF 653 kb)
Supplementary information, Figure S2
Cryo-EM analysis of the proteasome. (PDF 1343 kb)
Supplementary information, Figure S3
Work flow for the processing of the high-resolution proteasome-ADP-AlFx cryo-EM data. (PDF 498 kb)
Supplementary information, Figure S4
Pseudo atomic models of proteasome 19S RP subunits fitted into the cryo-EM map of proteasome-ADP-AlFx. (PDF 653 kb)
Supplementary information, Figure S5
Cryo-EM maps of yeast proteasome in the presence of ATP and conformation comparison with related previous cryo-EM maps. (PDF 1084 kb)
Supplementary information, Figure S6
Conformation comparison of our proteasome-ADP-AlFx map with the previously reported cryo-EM proteasome maps. (PDF 826 kb)
Supplementary information, Figure S7
The AAA-ATPase ring conformational change between proteasome-ADP-AlFx and the resting state structures. (PDF 988 kb)
Supplementary information, Figure S8
AAA-ATPase subunit interaction interface analysis and conformational changes of the AAA-ATPase ring from proteasome-ATPγS to proteasome-ADP-AlFx. (PDF 774 kb)
Supplementary information, Figure S9
Conformational changes of the AAA-ATPase subunits from the substrate-engaged state to the ADP-AlFx state. (PDF 544 kb)
Supplementary information, Figure S10
Conformational changes of Rpn10 and Rpn11 between proteasome-ATPγS and proteasome-ADP-AlFx. (PDF 871 kb)
Supplementary information, Table S1
Statistics of cryo-EM data collection and refinement. (PDF 74 kb)
Supplementary information, Movie S1
Comparison of our proteasome-ADP-AlFx map with the previous proteasome structures confirms that our proteasome-ADP-AlFx adopted a new conformation. (MOV 21868 kb)
Supplementary information, Movie S2
Base region conformational changes between proteasome-ATPγS and proteasome-ADP-AlFx, revealing the most prominent difference to be for Rpt2 in the AAA-ATPase ring and the origin of the gaps next to Rpt2. (MOV 18840 kb)
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Ding, Z., Fu, Z., Xu, C. et al. High-resolution cryo-EM structure of the proteasome in complex with ADP-AlFx. Cell Res 27, 373–385 (2017). https://doi.org/10.1038/cr.2017.12
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DOI: https://doi.org/10.1038/cr.2017.12
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