The mycobacterial proteasome – intramolecular communication between the inter- and AAA+

The bacterial proteasome 20S core particle (CP), typically comprising two homo-heptameric beta rings stacked between two homo-heptameric alpha rings, is controlled by a homo-hexameric AAA+ protein that acts as a regulatory particle. This regulatory particle (termed Mpa in mycobacteria) mediates initial binding of Pup (a ubiquitin analog), thus facilitating translocation of the pupylated substrate into the proteolytic chamber of the 20S CP18 (Fig. 1). Mpa comprises several distinctive domains. A coiled-coil domain close to the protein N-terminus performs initial Pup binding, in a manner that positions Pup N-terminal tail in the center of the Mpa AAA+ ring. Following this tethering step, Pup engagement by the AAA+ domain occurs, leading to substrate unfolding and translocation into the 20S CP. Common to all proteasome regulatory particle, a ring of OB (oligosaccharide binding) domain is found between the AAA+ domain and the N-terminal domain(s). In Mpa, two stacks of OB domains are found, together termed Inter-domain. Apparently, OB domains serve as pore forming proteasome subunits that allow substrate passage from the N-terminal domain(s) to the AAA+ domain. It remained unclear, however, whether OB domains play a more active role in the RP substrate processing activity. We find that in Mpa, the inter-domain critically affects the AAA+ domain ability to engage Pup. Indeed, single AA mutations in the inter-domain were found to abort engagement, while hardly affecting other Mpa activities. These findings suggest that the inter- and AAA+ domains interact at the molecular level, in a manner that facilitates their coordinated activity.