LONP1 structure overview.

A) Overview of LONP1 structural elements, labeled and colored by domain. The NTDGD and CCD are colored purple, the NTD3H and AAA+ are colored dark blue, and the PD are colored light blue. B) The conformation and structural elements that define the inactive open-form of LONP1 (LONP1OFF). The ATPase and protease domains adopt a left-handed open lock washer conformation, and all the ATPase domains are bound to ADP (white). The protease domains adopt an asymmetric conformation that positions an inhibitory 310 -helix (red) in the proteolytic active site cleft. C) The conformation and structural elements that define the active closed-form of LONP1 (LONP1ENZ). Substrate (orange) is bound by the NTD, which then threads through the CCD pore, where it is coordinated by the ATPase pore loops (white). The ATPase domains adopt a right-handed helical conformation that is competent for ATP (magenta) binding and hydrolysis. The PDs adopt a C6-symmetric conformation, resulting in a rearrangement of the inhibitory 310 -helix to form the proteolytic substrate binding site. D) Top view of the NTD showing the dimer of trimer arrangement. E) The top three NTD domains are removed to show the CCD pore with bound substrate (orange).

Structural overview of LONP1C3 state.

A) Density for the LONP1C3 map is shown with the asymmetric unit colored in shades of blue (down conformation) or green (up conformation) by domain. The color shades denote domain architecture (light is PD, medium is AAA+ and NTD3H, and dark is CCD). B) Overview of the LONP1C3 atomic model with key areas of regulation highlighted by the grey planes. The perspective eye provides the viewing angle for panels C-E. C) Conformational state of the CCD subdomain in LONP1C3. Residues H391 and L395 form a tightly packed core that prohibits entrance through the pore. D) The conformational state of the NTD3H subdomains of the three downward positioned subunits. E) Alternating up (green) and down (blue) pore loop conformations of the ATPase domains. F) The down-conformation ATPase active site bound to ADP. G) The up-conformation ATPase active site bound to ADP. H) Structural overlay of the up-conformation ATPase subunit (light green) with a LONP1ENZ ATP-bound subunit (white) demonstrating the similarity in active site conformation between the two nucleotide states.

ATPase and FITC-casein activity assays.

A) Basal and stimulated (with 10 µM casein) ATPase rates of wt and mutants normalized to wt activity. B) FITC-casein degradation rates of wt LONP1 and mutants normalized to wt activity. All assays were repeated in biological triplicate and error bars represent standard deviation of the mean.

ATPase kinetics wt and D449A/H451A double mutant.

Michaelis-Menten Kinetics of wt LONP1 and D449A/H451A ATP-ase activity. Table below graphical data provides catalytic parameters for wt and D449A/H451A double mutant.

Substrate dependence of C3-state.

A) Comparison of LONP1C3 conformational states across datasets of LONP1 degrading either StAR, TFAM, or β-casein. The LONP1-casein structure was obtained after applying symmetry expansion followed by 3D-classifiction, whereas LONP1-StAR and LONP1-TFAM were readily identified using standard processing pipelines (Figs S1, S6). Binarization threshold was set to 0.13 for all volumes. B) Gel-based degradation assay of StAR and TFAM with either wt LONP1 or the D449A mutant. The D449A mutant presents reduced degradation of StAR and TFAM despite wt activity levels against unstructured β-casein. Gel-based assays were repeated in duplicate. C) Substrate-dependent ATPase stimulate rates of wt LONP1, D449A, and D449A/H451A. Results indicate that the relative-to-wt ATPase stimulation is substrate dependent with mutations that destabilize LONP1C3. ATPase stimulation assays were repeated in biological triplicate and error bars represent standard deviation of the mean. D) Proposed mechanism where LONP1C3 is an on-pathway intermediate between the LONP1OFF and LONP1ENZ.

Structural overview of LONP1 processing intermediates.

A) Side view of LONP1C2 emphasizing the three-subunit right-handed ATPase spiral, with yellow at the bottom and light blue at the top. B) Top view of LONP1C2 demonstrating the C2-symmetry axis and nucleotide state of each ATPase domain. C) Cutaway and close-up view of the pore loops of the rear three-subunit right-handed spiral. D) Side-view of LONP1INT4 showing the four-subunit right-handed spiral. E) Top view of LONP1INT4 showing the more open central pore relative to LONP1C2 and nucleotide state of each ATPase domain. F) Cutaway and close-up view of the pore loops of the rear ATPase domains in LONP1INT4 forming a four-subunit right-handed spiral. G) Proposed order of conformational arrangements to transition from LONP1C3 to LONP1ENZ.

Cryo-EM data collection, image analysis, refinement, and statistics.

LONP1-StAR-ATP processing workflow.

Representative motion-corrected, dose-weighted cryo-EM micrograph, 2D averages, and image analysis pipeline used to determine final reconstructions. In the lower right the viewing distribution plot is shown, and underneath the global FSC and map-to-model FSC is overlaid on the directional resolution histogram from the 3DFSC server52, alongside the local resolution map of the final reconstruction, calculated using cryoSPARC.

Comparison of CCD in LONP1C3 and substrate-bound LONP1.

A) and B) Show top-view surface-representations comparing LONP1C3 and LONP1ENZ, respectively. C) and D) compare the approximate CCD dimensions of LONP1C3 and LONP1ENZ, respectively. E) and F) compare the angular pitch between CCD helices in LONP1C3 and LONP1ENZ, respectively.

Processing workflow for LONP1OFF from LONP1-StAR-ATP dataset.

This processing workflow was continued using the K2 “open” particles from the first heterogeneous refinement step shown in Fig. S1.

LONP1C3 and LONP1ENZ docked into LONP1OFF CCD density.

The atomic models of the CCD from the LONP1C3 and LONP1ENZ structures were rigid-body fit into the LONP1OFF CCD density, showing a greater similarity to LONP1C3.

Substrate structure and stability predictions.

AlphaFold2 was used to generate three-dimensional coordinates of StAR, TFAM and β-casein. The plDDT scores are mapped on top of each structure to demonstrate confidence in the predicted model. The instability index is also provided for each protein, which was calculated using ExPASY Prot Param tool.

Processing workflow for A) LONP1+TFAM+ATP and B) LONP1+Casein+ATP.

Processing workflow for LONP1C2, LONP1INT3, LONP1INT4.

Particles from the C1 N. U. Refinement of the LONP1+TFAM workflow in Fig. S6 were further processed to identify three intermediate conformations. For each reconstruction, the local resolution calculated using cryoSPARC is shown, as well as the global FSC and map-to-model FSC overlaid on the directional resolution histogram from the 3DFSC server. The corresponding viewing distribution plot is shown below.

LONP1C2 structural analysis.

A) Five intermediates placed in expected transition order from LONP1C3 to LONP1ENZ. LONP1C2 is proposed to represent the initial breaking of C3 symmetry and formation of symmetric half-spirals. B) LONP1C2 reconstruction colored by subunit position from lowest to highest (yellow, green, blue). C) LONP1ENZ docked into LONP1C2 density to demonstrate that the three bottom subunits of LONP1ENZ approximate the spiral forming in LONP1C2. An overlay of the pore loops of these positions is provided on the right hand side where the pore loops from LONP1ENZ are rendered in light grey and the pore loops of LONP1C2 follow their conventional coloring scheme from lowest to highest (yellow, green, blue). The orange substrate is derived from the LONP1ENZ model for reference. D) Pore loop density for LONP1C2. E) LONP1C3 coordinates docked into LONP1C2 to demonstrate that these conformations are somewhat similar and that four out of six subunits in LONP1C2 also have an alternating up and down arrangement. A cartoon overlay of the pore loops of LONP1C2 and LONP1C3 is provided for reference. The up and down subunits of LONP1C3 are colored in light and dark grey, respectively. LONP1C2 follows its canonical color convention denoting subunits from lowest to highest position in the spiral (yellow, green, blue). The overlay demonstrates that two subunits of LONP1C3 swapping position (up, down to down, up) would lead to the formation of a LONP1C2-like arrangement. F) The nucleotide density of the three subunits forming the asymmetric unit of LONP1C2, demonstrating that like LONP1C3, LONP1C2 is completely occupied by ADP.

Comparison of LONP1C2, LONP1INT3, and LONP1INT4 reconstructions.

Top and side views of LONP1C2, LONP1INT3, and LONP1INT4 demonstrating the progressive establishment of the fourth subunit (dark blue) in the asymmetric right-handed spiral seen in LONP1INT3 and LONP1INT4. Subunits are colored to show continuity in the progression from LONP1C2 to LONP1INT4.

Cryo-EM density for LONP1INT4 pore loops.

Stick representations of the pore loop residues are shown, with corresponding cryoEM density shown as a semi-transparent surface.

Nucleotide density for each subunit of LONP1INT4.

NTD3H as an allosteric signal integrator.

A) An overlay of the AAA+ domains of the up (blue) and down (green) conformation of LONP1C3 to demonstrate the overall architecture of the AAA+ domain and highlight regions with conformational differences between the conformers. The subdomains of LONP1 monomer are AAA+ large domain (AAALD), AAA+ small domain (AAASD), and protease domain (PD). The zoomed-in view provides a closer look at the AAALD demonstrating that the majority of the differences in conformation between the up and down positions in LONP1C3 are in the NTD3H, pore loops (outlined in red), and structurally associated regions. B) A more focused view of the AAALD highlighting key structural elements (NTD3H, pore loop, and presensor 1 β-hairpin (P1βH). A zoomed in view demonstrates the hydrophobic interface connecting these three regions into a unit. This network would allow conformational changes in the NTD during substrate recruitment to be communicated to key structural elements of the AAA+ via the NTD3H subdomain.