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 α and 14 β, arranged in four stacked seven-member rings (α7β7β7α7). While details of CP assembly vary across species, the final step in the assembly process is universally conserved: two half proteasomes (HPs; α7β7) 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 β subunit, which is autocatalytically cleaved off after CP formation, plays a key role in regulating this separation of timescales. However, the detailed molecular mechanism of how the propeptide achieves this regulation is unclear. In this work, we used molecular dynamics 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. Furthermore, we found that a propeptide mutant that dimerizes extremely slowly is essentially always in the nondimerizable state, while the wild-type rapidly transitions between the two. Based on these simulations, we designed a propeptide mutant that favored the dimerizable state in molecular dynamics simulations. In vitro assembly experiments confirmed that this mutant dimerizes significantly faster than wild-type. 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.

Proteasomes are multi-subunit protease complexes found in all domains of life. The maturation of the core particle (CP), which harbors the active sites, involves dimerization of two half CPs (HPs) and an autocatalytic cleavage that removes β propeptides. How these steps are regulated remains poorly understood. Here, we used the Rhodococcus erythropolis CP to dissect this process in vitro. Our data show that propeptides regulate the dimerization of HPs through flexible loops we identified. Furthermore, N-terminal truncations of the propeptides accelerated HP dimerization and decelerated CP auto-activation. We identified cooperativity in autocatalysis and found that the propeptide can be partially cleaved by adjacent active sites, potentially aiding an otherwise strictly autocatalytic mechanism. We propose that cross-processing during bacterial CP maturation is the underlying mechanism leading to the observed cooperativity of activation. Our work suggests that the bacterial β propeptide plays an unexpected and complex role in regulating dimerization and autocatalytic activation.