Structural Insights into Mdn1, an Essential AAA Protein Required for Ribosome Biogenesis

Abstract

SUMMARY Mdn1 is an essential AAA (ATPase Associated with various Activities) protein that removes assembly factors from distinct precursors of the ribosomal 60S subunit. However, Mdn1’s large size (~5000aa) and its limited homology to other well-studied proteins have restricted our understanding of its remodeling functions. Here, we present structures for S. pombe Mdn1 in the presence of AMPPNP at up to ~4Å, or ATP plus Rbin-1, a chemical inhibitor, at ~8Å. These data reveal that Mdn1’s MIDAS domain is tethered to its ring-shaped AAA domain through an ~20nm long structured linker and a flexible ~500aa Asp/Glu-rich motif. We find that the MIDAS domain, which also binds other ribosome-assembly factors, docks onto the AAA ring in a nucleotide state-specific manner. Together, our findings reveal how conformational changes in the AAA ring can be directly transmitted to the MIDAS domain and thereby drive the targeted release of assembly factors from ribosomal 60S-subunit precursors.

The role of the adaptor protein CbbO in the Rubisco activation system of chemoautotrophic bacteria

“…Taken together, the results in this chapter support the hypothesis that the structural ensemble of the CbbQO-type Rubisco activases resemble that of other Helix-2-insert clade of AAA+ proteins such as Mdn1 where the adaptor CbbO extends from the AAA+ ring and folds back upon itself to enable the tethered C-terminal VWA domain to dock on the AAA+ ring (Chen et al, 2018). This raises the question if the mechanism of action is similar as well.…”

“…(Midasin) and Rea1 resemble the structure of CbbQO with a hexameric head domain from which an elongated flexible tail carrying the MIDAS domain protrude (Chen et al, 2018;Sosnowski et al, 2018). In the reported structures, the linker stems from the outer edge of the AAA+ ring and the C-terminal VWA domain docks onto the AAA+ ring in a nucleotide state-specific manner.…”

“…In support of this, a direct interaction between the density occupying the central cavity of the CbbQ3 cup, believed to be the VWA domain and Rubisco can be seen in our map. Further, both the yeast ribosome maturation factors Rea1 and Mdn1 (Midasin) VWA domain is docked onto the AAA+ ring and is in close contact with the substrate (Chen et al, 2018;Sosnowski et al, 2018).…”

“…The N-terminal region (residues 1-444) of CbbO is believed to interact with CbbQ forming a hetero-oligomer with a stoichiometry of CbbQ6O1 (Tsai et al, 2015). Thus, the subunit organization of CbbQO has been proposed to resemble the largest yeast protein, ribosome maturation protein Rea1 or Midasin, which presents as a hexameric head domain from which an elongated flexible tail carrying the VWA domain protrude (Barrio-Garcia et al, 2016;Chen et al, 2018;Sosnowski et al, 2018). However, the insolubility of CbbO isoforms in E. coli unless co-expressed with their respective CbbQ isoforms poses as a challenge in biochemically characterizing CbbO and understanding its role in this Rubisco activation system (Tsai et al, 2015).…”

“…The other three adjacent subunits are clearly visible. Importantly, the density in the central cavity of CbbQ is reminiscent of the VWA domain docking on the AAA+ ring of the yeast ribosome maturation factor Rea1 and Mdn1 (Midasin), suggesting a similar arrangement(Chen et al, 2018;Sosnowski et al, 2018).…”