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bioRxiv · 10.1101/2022.02.04.479176

Understanding the Separation of Timescales in Rhodococcus erythropolis Proteasome Core Particle Assembly

Itagi, P.·Kante, A.·Lagunes, L.·Deeds, E. J.
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Abstract

The 20S proteasome Core Particle (CP) is a molecular machine that is a key component of cellular protein degradation pathways. Like other molecular machines, it is not synthesized in an active form, but rather as a set of subunits that assemble into a functional complex. The CP is conserved across all domains of life and is composed of 28 subunits, 14 and 14 {beta}, arranged in four stacked 7-member rings (7{beta}7{beta}77). While details of CP assembly vary across species, the final step in the assembly process is universally conserved: two half proteasomes (HP: 7{beta}7) dimerize to form the CP. In the bacterium Rhodococcus erythropolis, experiments have shown that the formation of the HP is completed within minutes, while the dimerization process takes hours. The N-terminal propeptide of the {beta} subunit, which is autocatalytically cleaved off after CP formation, plays a key role in regulating this separation of time scales. However, the detailed molecular mechanism of how the propeptide achieves this regulation is unclear. In this work, we used Molecular Dynamics (MD) simulations to characterize HP conformations and found that the HP exists in two states: one where the propeptide interacts with key residues in the HP dimerization interface and likely blocks dimerization, and one where this interface is free. We found that a propeptide mutant that dimerizes extremely slowly is essentially always in the non-dimerizable state, while the WT rapidly transitions between the two. Based on these simulations, we designed a propeptide mutant that favored the dimerizable state in MD simulations. In vitro assembly experiments confirmed that this mutant dimerizes significantly faster than WT. Our work thus provides unprecedented insight into how this critical step in CP assembly is regulated, with implications both for efforts to inhibit proteasome assembly and for the evolution of hierarchical assembly pathways.

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Itagi, P., Kante, A., Lagunes, L., Deeds, E. J.. 2022-02-05. Understanding the Separation of Timescales in Rhodococcus erythropolis Proteasome Core Particle Assembly. https://doi.org/10.1101/2022.02.04.479176

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