Cryo-EM Reveals Regulatory Mechanisms Governing Substrate Selection and Activation of Human LONP1

Reviewer #1 (Public review):

The remodeling of macromolecular substrates by AAA+ proteins is an essential aspect of life at the molecular scale, and understanding conserved and divergent features of substrate recognition across the AAA+ protein family remains an ongoing area of research. AAA+ proteins are highly modular and typically combine N-terminal recognition domain(s) with ATPase domain(s) to recognize and unfold some macromolecular target, such as dsDNA or protein substrates. This can be coupled to activity by additional C-terminal domains that further modify the substrate, such as a protease domain that hydrolyzes the extended, unstructured protein chain that emerges from the ATPase domain during substrate processing.

This work focuses on one such AAA+ protease, LONP1. LONP1 is an essential AAA+ protein involved in mitochondrial proteostasis, and disruption of its function in vivo has serious developmental consequences. This work explores the processing of two new mitochondrial protein substrates (StAR, TFAM) by LONP1 and presents new conformational states of LONP1 with closed configurations and no substrate threaded through the ATPase pores. The quality of the reconstructions and models is very good. Critically, one of these states (LONP1C3) has a completely occluded ATPase pore from the N-terminal side of the ATPase ring, where three of the six NTDs/CCDs interact tightly to form a C3-symmetric substructure preventing substrate ingress. The authors note several key interactions between amino acids forming these substructures, and perform ATPase assays on mutant LONP1 proteins to determine hydrolysis rates in the absence or presence of substrate. These patterns are recapitulated in casein disassembly assays as well. Based on these results, the authors note that the mutants have differential effects depending on the "foldedness" of the substrate, and surmise that disruption of the C3-symmetric substructure from the EM experiments is responsible for these effects - an intriguing idea. In addition to the C3 state, the authors observe additional intermediates which they place on the same conformational coordinate. One such structure is the LONP1C2 state with two splits, hinting at a conformational transition from LONP1C3 to the closed/active state.

Taken together, these results form the basis of an interesting story. However, I feel that more experimentation and analysis are needed to address several key points, or that the conclusions should be toned down. First and foremost, I note that while the hypothesis that the LONP1C3 state is a critical step in recognizing substrate "foldedness" is an interesting one, the claim is made solely on the basis of biochemical experiments with mutant LONP1, and that there is no substrate density associated with LONP1C3. In the absence of substrate density and/or structural data for the mutants, this seems like a very strong claim. More generally, the manuscript invokes the conformational landscape of LONP1C3 in multiple instances, but no such landscape is presented to show how LONP1C3 and the other states are quantitatively linked. Finally, I note the prevalence of ADP-only active sites in these intermediates, and am concerned that this might be related to the depletion of ATP under the on-grid reaction conditions. The inclusion of an ATP regeneration system may be a useful way to ensure that ATP/ADP concentrations are more physiological and that excessive ADP will not bias the conformations of the ring systems.

In summary, I believe this manuscript is exciting but would benefit from a paring back of claims, or the inclusion of some additional data to fill in some of the conceptual gaps outlined above.

Reviewer #2 (Public review):

This paper by Mindrebo et al. reveals multiple novel conformations of the human LONP1 protease. AAA+ proteases, like LONP1, are needed for maintaining proteostasis in cells and organelles. While structures of fully active (closed) and fully inactive (open) conformations of LONP1 are now established, the dynamics between these states and how changes in conformations may contribute to or be triggered by substrates and nucleotides are unclear. In this work, the authors characterize a novel C3-symmetric state of LONP1 bound to TFAM (a native substrate), suggesting that this C3-state is an intermediate in the open to closed cycle, and make mutations to test this model biochemically. Deeper inspection of their TFAM-bound LONP1 dataset reveals additional conformations, including a C2-symmetric and two asymmetric intermediates. All these conformations are synthesized by the authors to propose a model for how LONP1 transitions from an inactive OFF state to an active ENZ state. There are clear, interesting structural aspects to this work, revealing alternate conformations to shed light on the dynamics of LONP1. However, some of the conclusions interpret well beyond the scope of the experiments shown, and this is discussed below.

Overall, there are two major comments with the work as written that, if addressed, would make the results more compelling. First, the order of events and existence of intermediate states is primarily from static structural snapshots and fitting these structures to a possible mechanism. It would be ideal to have some biochemical or kinetic data supporting these steps and the existence of these intermediates. For example, the model is that the C3-state is an ADP-bound intermediate that blocks access and acts as a checkpoint for progression to the ENZ state of LONP1. The major evidence for this comes from a mutation (D449A) that fails to degrade TFAM as well as StAR or casein, which is taken as evidence that failure to form the C3 state reduces the ability to degrade more 'folded' substrates. A prediction of this model would be that destabilizing TFAM through mutation should improve D449A degradation. Ideally, other measures of conformational changes, such as FRET or HDX-MS, could be used to visualize this C3-state in unmutated LONP1 during the process of substrate engagement and degradation. At a minimum, using ATP hydrolysis as a proxy for forming the ENZ state and the assumption that different substrates will differentially promote formation of the C3-state means that measuring ATP hydrolysis of wt LONP1 with different substrates will be informative.

The second major comment is that the primary evidence for the importance of the C3 state is a mutation (D449A) that, based on the cryoEM structure, is incompatible with this conformation but should not affect any other state. A concern that arises is whether this mutation is doing more than simply destabilizing the C3 state and affecting substrate recognition/enzymatic activity in some other manner. To address this point, the authors could perform cryoEM characterization of the D449A mutant, which should show reduced or no presence of the C3-state, but still an intact ability to form the closed ENZ state.

Reviewer #3 (Public review):

Summary:

The AAA+ protease LON1P is a central component of mitochondrial protein quality control and has crucial functions in diverse processes. Cryo-EM structures of LON1P defined inactive and substrate-processing active states. Here, the authors determined multiple new LON1P structural states by cryo-EM in the presence of diverse substrates. The structures are defined as on-pathway intermediates to LON1P activation. A C3-symmetry state is suggested to function as a checkpoint to scan for LON1P substrates and link correct substrate selection to LON1P activation.

Strengths:

The determination of multiple structures provides relevant information on substrate-triggered activation of LON1P. The authors support structural data by biochemical analysis of structure-based mutants.

Weaknesses:

How substrate selection is achieved remains elusive, also because substrates are not detectable in the diverse structures. It also remains in parts unclear whether mutant phenotypes can be specifically linked to a single structural state (C3). Some mutant phenotypes appear complex and do not seem to be in line with the model proposed.