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bioRxiv · 10.1101/2020.12.22.423932

Deep manifold learning reveals hidden dynamics of proteasome autoregulation

Wu, Z.·Zhang, S.·Wang, W. L.·Ma, Y.·Dong, Y.·Mao, Y.
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Abstract

The 26S proteasome regulates proteostasis and myriad cellular processes. Here we reconstitute the hidden dynamics of human 26S proteasome during protein degradation using cryo-EM data-driven deep learning, which identified 64 proteasomal conformers. These conformers revealed substrate-dependent conformational entanglement of two 19S regulatory particles in the doubly capped holocomplexes. The rate-limiting step in single-nucleotide exchange dynamics of the AAA-ATPase motor is ATPase re-engagement with the substrate, while the rate-limiting step in proteasomal degradation is the initiation of substrate unfolding by the AAA-ATPase motor following deubiquitylation. Nine rare conformers delineated novel ubiquitin-binding sites on the RPN2 and 5 subunits, along with a ubiquitylation site on RPN10, which were verified using site-directed mutagenesis and liquid chromatography-tandem mass spectrometry. Opening of the 20S core particle gate was found to upregulate proteolytic activity by allosterically promoting nucleophilic attack on the scissile peptide bond. Our systematic analyses illuminate a grand hierarchical allostery underlying ubiquitin-mediated proteasome autoregulation.

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Wu, Z., Zhang, S., Wang, W. L., Ma, Y., Dong, Y., Mao, Y.. 2020-12-22. Deep manifold learning reveals hidden dynamics of proteasome autoregulation. https://doi.org/10.1101/2020.12.22.423932

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