The mycobacterial proteasomal ATPase Mpa forms a gapped ring to engage the 20S proteasome

Yanting Yin,Amanda Kovach,Hao-Chi Hsu,K Heran Darwin,Huilin Li

doi:10.1016/j.jbc.2021.100713

Yanting Yin, Amanda Kovach + Show 3 more

Open Access

https://doi.org/10.1016/j.jbc.2021.100713

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Abstract

Although many bacterial species do not possess proteasome systems, the actinobacteria, including the human pathogen Mycobacterium tuberculosis, use proteasome systems for targeted protein removal. Previous structural analyses of the mycobacterial proteasome ATPase Mpa revealed a general structural conservation with the archaeal proteasome-activating nucleotidase and eukaryotic proteasomal Rpt1–6 ATPases, such as the N-terminal coiled-coil domain, oligosaccharide-/oligonucleotide-binding domain, and ATPase domain. However, Mpa has a unique β-grasp domain that in the ADP-bound crystal structure appears to interfere with the docking to the 20S proteasome core particle (CP). Thus, it is unclear how Mpa binds to proteasome CPs. In this report, we show by cryo-EM that the Mpa hexamer in the presence of a degradation substrate and ATP forms a gapped ring, with two of its six ATPase domains being highly flexible. We found that the linkers between the oligonucleotide-binding and ATPase domains undergo conformational changes that are important for function, revealing a previously unappreciated role of the linker region in ATP hydrolysis–driven protein unfolding. We propose that this gapped ring configuration is an intermediate state that helps rearrange its β-grasp domains and activating C termini to facilitate engagement with proteasome CPs. This work provides new insights into the crucial process of how an ATPase interacts with a bacterial proteasome protease.

Highlights

Unfolded, and delivered into 20S core particle (CP) for degradation by a regulatory structure that includes at its base a heterohexameric ATPase ring [1, 8]
prokaryotic ubiquitin-like protein (Pup) is a mostly disordered small protein (64 amino acids in Mycobacterium tuberculosis (Mtb)) ending in Gln (Q), which needs to be converted to Glu (E) by the deamidase of Pup before it can be covalently linked to a lysine in a target protein by the ligase proteasome accessory factor (Paf) A in an ATP-dependent mechanism [11]
From a 4-Å resolution cryo-EM structure in the presence of a pupylated substrate, we show that an Mpa hexamer binds one ATP and two ADP molecules to form a gapped-ring structure that can better interact with a 20S CP

Summary

Results

Pupylated substrate binding to Mpa stimulates ATPase activity in vitro In Escherichia coli, we separately produced and purified the full-length Mtb Mpa and Pup–malonyl CoA-acyl carrier protein transacylase (FabD), in which Pup was translationally fused to the N terminus of FabD. Mpa hexamers in solution, whether in the presence of ATP, AMP-PNP, or ATPγS, seem to have all six coiled-coil domains largely flexible, with all six double OB domains forming the stable double OB ring, but with the AAA domains of two Mpa protomers being highly flexible and invisible in the 3D reconstructions. An Mpa hexamer in the presence of 5 mM ATP is in a mixed nucleotide state, bound to both ATP and ADP This gapped flexible ring architecture contrasts with our previous crystal structures that show six AAA domains forming a stable and closed flat ring in the fully ADP-bound state. Superimposing the shared double OB ring of the cryo-EM structure and the ADP-bound crystal structure revealed detailed changes of individual AAA domains in these two states (Fig. 4B).

F Bottom view of AAA ring

B Mpa in ADP-bound closed ring form

Discussion

Experimental procedures