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 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 underlying molecular mechanisms governing 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. Unlike bacterial Lon, human LONP1 contains a second spiral staircase within its ATPase domain that engages substrate as it is translocated toward the proteolytic chamber. Intriguingly, and in contrast to its bacterial ortholog, substrate binding within the central ATPase channel of LONP1 alone is insufficient to induce the activated conformation of the protease domains. To successfully induce the active protease conformation in substrate-bound LONP1, substrate binding within the protease active site is necessary, which we demonstrate by adding bortezomib, a peptidomimetic active site inhibitor of LONP1. These results suggest LONP1 can decouple ATPase and protease activities depending on whether AAA+ or both AAA+ and protease domains bind substrate. Importantly, our structures provide a molecular framework to define the critical importance of LONP1 in regulating mitochondrial proteostasis in health and disease.

Molecular basis for ATPase-powered substrate translocation by the Lon AAA+ protease

“…However, the simple one-step transition model is not supported by recent cryo-EM structures of substrate-bound AAA+ proteins with the ATPase domains organized in spiral staircase arrangement around a centrally positioned substrate polypeptide chain ( 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 ). These structures support a processive rotary mechanism, which was also suggested by recent cryo-EM structures of human and Yersinia pestis Lon proteases ( 23 , 24 ). However, in these works the bacterial Lon used for reconstruction was a Walker-B mutant; the structures of human and bacterial Lon show different engagement with the bound substrate from the six protomers, with different sets of nucleotide-binding states ( 23 , 24 ).…”

“…These structures support a processive rotary mechanism, which was also suggested by recent cryo-EM structures of human and Yersinia pestis Lon proteases ( 23 , 24 ). However, in these works the bacterial Lon used for reconstruction was a Walker-B mutant; the structures of human and bacterial Lon show different engagement with the bound substrate from the six protomers, with different sets of nucleotide-binding states ( 23 , 24 ).…”

“…The substrate-bound structure forms an asymmetric close ring. Interestingly, recent cryo-EM structures of substrate-free Lon show an open spiral structure, where the side opening potentially allows access of the pore loops to substrates from outside ( 11 , 23 , 24 ). Nevertheless, the substrate Ig2 is more likely to be translocated from the top of the AAA+ ring, where the substrate-binding N-terminal domain, essential for recognition and proteolysis of Ig2 ( 25 ), is located.…”

Complete three-dimensional structures of the Lon protease translocating a protein substrate

“…4B ). The protomers P5 and P6 serve as two ascending nonengaging “seam” protomers, an arrangement similar to human mitochondrial Lon but not Yersinia pestis Lon ( 19 – 21 ). In Con1, the P1 substrate-engaging protomer is an Ac-protomer, with an acute-angled LH, whereas in Con2, the P1 protomer is an Ob-protomer; therefore, Con1 and Con2 may be regarded as two sequential conformational states in the ATP-hydrolysis cycle, which proceeds counterclockwise in each of the six protomers ( Fig.…”

Processive cleavage of substrate at individual proteolytic active sites of the Lon protease complex

“…This substrate binding mode has not been observed previously for bacterial Lon ( 19 , 20 ). In the recent structure of human Lonp1, the bound substrate is similarly gripped by pore-loop-1 from five protomers; however, the bound polypeptide is engaged only by pore-loop-2 from two protomers ( 21 ).…”