Distinct quaternary structures of the AAA+ Lon protease control substrate degradation

Lon is an ATPase associated with cellular activities (AAA+) protease that controls cell division in response to stress and also degrades misfolded and damaged proteins. Subunits of Lon are known to assemble into ring-shaped homohexamers that enclose an internal degradation chamber. Here, we demonstrate that hexamers of Escherichia coli Lon also interact to form a dodecamer at physiological protein concentrations. Electron microscopy of this dodecamer reveals a prolate structure with the protease chambers at the distal ends and a matrix of N domains forming an equatorial hexamer–hexamer interface, with portals of ∼45 Å providing access to the enzyme lumen. Compared with hexamers, Lon dodecamers are much less active in degrading large substrates but equally active in degrading small substrates. Our results support a unique gating mechanism that allows the repertoire of Lon substrates to be tuned by its assembly state.Protein quality control is vital under stress conditions that promote protein unfolding and aggregation. Escherichia coli Lon degrades many unfolded proteins (1–3) and also degrades folded proteins, including SulA (supressor of Lon protein) and the inclusion-body binding proteins A and B (IbpA and B) (4–6). In E. coli and many other bacteria, Lon is up-regulated under numerous stress conditions (7–10). In mitochondria, Lon helps combat oxidative stress (11–14), and human mitochondrial Lon was recently identified as a potential antilymphoma target (15). It is widely believed that a major role of Lon in all organisms is to degrade misfolded proteins (2, 10, 16).Lon subunits consist of an N domain, a central ATPase associated with cellular activities (AAA+) ATPase module, and a C-terminal peptidase domain. Although early reports suggested that Lon might be a tetramer (17), it is now clear that six subunits of the E. coli enzyme assemble into a hexamer with an internal degradation chamber accessible via an axial pore in the AAA+ ring (18, 19). Lon substrates are recognized, unfolded if necessary by ATP-dependent reactions mediated by the AAA+ ring, and then translocated through the pore and into the peptidase chamber for degradation (20).In many families of ATP-dependent proteases, the AAA+ unfolding/translocation ring and the self-compartmentalized peptidase are encoded by distinct polypeptides, which assemble into independent oligomers before interacting to form the functional protease (21, 22). For example, the ClpXP protease consists of AAA+ ClpX hexamers, which dock with the self-compartmentalized ClpP peptidase. This interaction suppresses the ATPase rate of ClpX and enhances the peptidase activity of ClpP (22). Lon activity cannot be controlled in this way because the ATPase and protease domains are always physically attached. Little is currently known about how Lon activity is regulated, although mutational studies show that the AAA+ and peptidase domains influence each other’s activities (23–25). In some cases, the function of the two domains also appears to be linked via allosteric communication mediated by substrate binding (26, 27).Here, we demonstrate that Lon forms dodecamers that equilibrate with hexamers at physiological concentrations. A structure determined by EM at low resolution reveals a unique protease architecture with the degradation chambers of each hexamer at opposite ends of a prolate ellipsoid. Near the equator of this structure, the arrangement of N domains creates portals, which could serve as entry sites for protein substrates. Formation of the dodecamer suppresses proteolysis of large but not small protein substrates, suggesting that the dodecamer uses a gating mechanism that allows the repertoire of Lon substrates to be tuned by its state of assembly.