Structures of the Human LONP1 Protease Reveal Regulatory Steps Involved in Protease Activation

Abstract

The human mitochondrial AAA+ protein LONP1 is a critical quality control protease involved in regulating diverse aspects of mitochondrial biology including proteostasis, electron transport chain activity, and mitochondrial transcription. As such, genetic or aging-associated imbalances in LONP1 activity are implicated in the pathologic mitochondrial dysfunction associated with numerous human diseases. Despite this importance, the molecular basis for LONP1-dependent proteolytic activity remains poorly defined. Here, we solved cryo-electron microscopy structures of human LONP1 to reveal the molecular mechanism of substrate proteolysis. We show that, like bacterial Lon, human LONP1 adopts both an open and closed spiral staircase orientation dictated by the presence of substrate and nucleotide. However, unlike bacterial Lon, human LONP1 contains a second spiral staircase within its ATPase domain that engages substrate to increase interactions with the translocating peptide as it transits into the proteolytic chamber for proteolysis. Further, we show that substrate-bound LONP1 includes a second level of regulation at the proteolytic active site, wherein autoinhibition of the active site is only relieved by the presence of a peptide substrate. Ultimately, our results define a structural basis for human LONP1 proteolytic activation and activity, establishing a molecular framework to understand the critical importance of this protease for mitochondrial regulation in health and disease.

Catalytic cycling of human mitochondrial Lon protease

“…The substructure of the hexameric assembly of AAA+ and protease domains from Yersinia pestis indicated it is activated by inversion of the helical arrangement of its AAA+ domains (18). This and a similar study of human LonP1 (19) showed that protease activation closes a lateral gap between two subunits around an unfolded, extended chain of the substrate protein. As a result, tyrosine-valine motifs located on the loops of the AAA+domain bind to the extended substrate protein strand in an axial tunnel.…”

“…Protease activity of bacterial Lon protease is activated by structural rearrangements around the catalytic S855/K898 dyad that are induced by the transition from the R-state to the P-state (18, 19). However, in our LonP1 structures, all individual R- and P-state proteolytic domains superimposed very well with RMS deviations of Cα-positions in the sub-Å range, and we did not observe full occlusion of the reactive S855 by other structural elements in any of the states, including D852, which has been identified as a regulatory element in bacterial Lon protease (21).…”

A dual allosteric pathway drives human mitochondrial Lon

“…During preparation of this manuscript, a cryo-EM structure of a shortened human Lon construct (115–959) in an inactive conformation was presented (Shin et al, 2021). In that case, only the ATPase domains and PDs were ordered and the PDs were in a staggered non-active conformation.…”

“…This contrasts with other AAA+ proteases such as ClpXP or the 26S proteasome (Baker & Sauer, 2012;Bard et al, 2018;Lee et al, 2011), in which the ATPase and PD domains are in separate chains and function as obligate heterooligomers. Six Lon monomers assemble into a barrel-shaped structure (Cha et al, 2010;Duman & Löwe, 2010;Kereïche et al, 2016;Vieux et al, 2013;Shin et al, 2020;Shin et al, 2021;Zhang et al, 2020). The LAN domains specifically recognize and capture fully or partially unfolded substrates (Tzeng et al, 2021;Wohlever et al, 2014), which are then threaded towards the barrel chamber for degradation using the chemical energy from ATP hydrolysis (Duman & Löwe, 2010;He et al, 2018;Li et al, 2005Li et al, , 2010Vieux et al, 2013).…”

“…Moreover, the LAN domains together with the neck region play a critical role in oligomerization (Kereïche et al, 2016). The unfolded substrate is threaded through the Lon barrel by two flexible loops in the AAA+ domain, namely pore loop 1 and 2, towards the protease domain at the bottom (Shin et al, 2020; Shin et al, 2021; Zhang et al, 2020). The protease active site is formed by a Ser-Lys dyad that is guarded by a loop (termed gate loop) that opens upon Lon hexamerization (Botos et al, 2004; Lin et al, 2016).…”